Coexistence of multiple peptides in small intensely fluorescent (SIF) cells of inferior mesenteric ganglion of the guinea pig

Summary Coexistence of peptides in the small intensely fluorescent cells was demonstrated by immunocytochemistry for met-enkephalin-Arg-Gly-Leu, vasoactive intestinal polypeptide, somatostatin, neuropeptide Y and dynorphin. In the extreme example, a single cell was immunoreactive to all 5 peptides examined. Four peptides coexisted in 8% and three peptides in 13% of SIF cells. In 10% of SIF cells no peptide immunoreactivity could be detected. The most prevalent peptide was met-enkephalin (in 46% of cells), then vasoactive intestinal polypeptide (45%), somatostatin (39%), neuropeptide Y (31%) and dynorphin (24%). Met-enkephalin and vasoactive intestinal polypeptide coexisted most commonly (25%).     

生长抑素mRNA与神经肽Y，催产素在大鼠前脑内的分布及其共存现象──地高辛标记c－RNA探针原位杂交与免疫组化联合法

本实验应用地高辛标记ｃＲＮＡ探针原位杂交组化和免疫组化联合法在同一切片上先后显示了生长抑素ｍＲＮＡ神经元和催产素神经元,生长抑素ｍＲＮＡ神经元和神经肽Ｙ神经元。结果表明生长抑素ｍＲＮＡ及神经肽Ｙ广泛地共存于大鼠的大脑新皮质,尾壳核,以及海马等处的神经元中。所有位于大脑新皮质,尾壳核处的神经肽Ｙ神经元均含有生长抑素ｍＲＮＡ,部分位于海马的神经肽Ｙ神经元含有生长抑素ｍＲＮＡ,而所有位于下丘脑弓状核,室周核的神经肽Ｙ神经元均不含有生长抑素ｍＲＮＡ;生长抑素ｍＲＮＡ与催产素虽然共同分布于下丘脑许多核区,但未见共存于同一神经元。本文对地高辛标记ｃＲＮＡ探针原位杂交组化以及它与免疫组化联合法的技术问题进行了讨论。     

Insulin deficiency is a specific stimulus to hypothalamic neuropeptide Y: a comparison of the effects of insulin replacement and food restriction in streptozocin-diabetic rats.

Untreated insulin-deficient diabetes causes hyperphagia and neuroendocrine disturbances that may be partly mediated by increased hypothalamic activity of neuropeptide Y (NPY), a potent central appetite stimulant. The metabolic signal that stimulates hypothalamic NPY is unknown. This study aimed to determine whether insulin deficiency or hyperglycemia was responsible. Regional hypothalamic NPY concentrations were compared in streptozocin-diabetic (STZ-D) rats rendered nearly normoglycemic by either insulin replacement or food restriction. Untreated STZ-D rats were hyperphagic and showed significantly increased (p less than 0.01) hypothalamic NPY concentrations in the arcuate nucleus and lateral hypothalamic area. Once-daily ultralente insulin injections corrected hypoinsulinemia and hyperglycemia, abolished hyperphagia, and normalized NPY concentrations in all hypothalamic regions. By contrast, food restriction effectively lowered glycemia without raising insulin levels. In these underfed diabetic rats, NPY concentrations rose further and were significantly higher than nondiabetic and untreated diabetic levels in most hypothalamic regions. We conclude that insulin deficiency is a major stimulus to hypothalamic NPY in STZ-D, whereas hyperglycemia may exert an inhibitory influence. These findings support the hypothesis that hypothalamic NPY responds to specific metabolic cues and is involved in regulating energy balance and conserving body weight.     

Peptides and neurotransmission in the central nervous system

Radioimmunoassays of brain extracts have shown that several peptides occur in high concentrations in the CNS. The releasing-factor peptides TRF, LRF, somatostatin, CRF and GRF have the highest concentration in the hypothalamic extracts. High levels of somatostatin, CCK octapeptide, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are found in cortical extracts. Substance P, CCK, NPY, and enkephalins are present in high concentrations in basal ganglia and mesolimbic areas. Pharmacological doses of these peptides result in several behavioural and vegetative effects. Immunocytochemical studies show that the CNS peptides are localised in neurones and in synaptic vesicles. In vitro studies with brain tissues show that peptides are capable of modifying the ongoing classical neurotransmission. In depressive patients several neuropeptides (CCK, CRF and NPY) have been shown to have low CSF levels. Patients dying of senile dementia have low cortical levels of somatostatin, CRF and substance P. In schizophrenic patients CCK peptides have shown to improve some symptoms. At present the therapeutic potentials of peptides are poorly known. More studies are required to understand their role in neurotransmission and related pathological states.     

Islet neuronal abnormalities associated with impaired insulin secretion in type 2 diabetes in the Chinese hamster.

This study examined the relationship between islet neurohormonal characteristics and the defective glucose-stimulated insulin secretion in genetic type 2 diabetic Chinese hamsters. Two different sublines were studied: diabetes-prone CHIG hamsters and control CHIA hamsters. The CHIG hamsters were divided into three subgroups, depending on severity of hyperglycemia. Compared to normoglycemic CHIG hamsters and control CHIA hamsters, severely hyperglycemic CHIG hamsters (glucose > 15 mmol/l) showed marked glucose intolerance during i.p. glucose tolerance test and 75% impairment of glucose-stimulated insulin secretion from isolated islets. Mildly hyperglycemic CHIG animals (glucose 7.2-15 mmol/l) showed only moderate glucose intolerance and a 60% impairment of glucose-stimulated insulin secretion from the islets. Immunostaining for neuropeptide Y and tyrosine hydroxylase (markers for adrenergic nerves) and for vasoactive intestinal peptide (marker for cholinergic nerves) revealed significant reduction in immunostaining of islets in the severely but not in the mildly hyperglycemic animals, compared to control CHIA hamsters. The study therefore provides evidence that in this model of type 2 diabetes in Chinese hamsters, severe hyperglycemia is accompanied not only by marked glucose intolerance and islet dysfunction but also by reduced islet innervation. This suggests that islet neuronal alterations may contribute to islet dysfunction in severe but not in mild diabetes.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

Unchanged hypothalamic neuropeptide Y concentrations in hyperphagic, hypoglycemic rats: evidence for specific metabolic regulation of hypothalamic NPY

Hypothalamic concentrations of neuropeptide Y (NPY), a potent central appetite stimulant, increase dramatically in food-restricted and insulin-deficient diabetic rats. This suggest that NPY may drive hyperphagia in these conditions, which are characterized by weight loss and insulin deficiency. To test the hypothesis that insulin deficiency and weight loss are specific stimuli to hypothalamic NPY, we measured NPY concentrations in individual hypothalamic regions in rats with hyperphagia caused by insulin-induced hypoglycemia. Groups of 8 male Wistar rats were injected with ultralente insulin (20-60 U/kg) to induce either acute hypoglycemia (7 h after a single injection) or chronic hypoglycemia (8 days with daily injections). In hypoglycemic rats, plasma insulin concentrations were increased 6- to 7-fold compared with saline-injected controls; food intake was significantly increased with acute and chronic hypoglycemia and weight gain was significantly increased in the chronically hypoglycemic group. NPY concentrations were measured by radioimmunoassay in 8 hypothalamic regions microdissected from fresh brain slices. NPY concentrations were not increased in any region in either acute or chronic hypoglycemia. NPY therefore seems unlikely to mediate hyperphagia in hyperinsulinemia-induced hypoglycemia, supporting the hypothesis that weight loss is a specific stimulus to hypothalamic NPY and that insulin deficiency may be the metabolic signal responsible.     

Neuropeptide Y is an inhibitor of neural function in the myenteric plexus of the guinea-pig ileum

Neuropeptide Y (NPY) reduced the resting tension of the myenteric plexus-longitudinal muscle preparation (MP-LM) of the guinea-pig ileum (GPI). NPY in a dose-dependent manner also reduced the neurally-mediated excitatory effect of cholecystokinin octapeptide (CCK8) sulfated form on this preparation. However, NPY, at the concentration used in the study, did not modify the effect of exogenous acetylcholine (ACh). All these features were also shared by other inhibitory peptides, like somatostatin (SOM) and the enkephalin derivative FK 33-824. The preparation developed a degree of tachyphylaxis to the inhibitory effect of NPY more rapidly than it did to SOM. Moreover, the inhibitory effect of neuropeptide Y was of longer duration than the one seen for somatostatin. A faster metabolic rate might account for the lower development of tachyphylaxis to somatostatin. The presence of the opioid antagonist naloxone did not alter the inhibitory features of NPY or SOM. Therefore, the involvement of any endogenous opioid in the action of these two inhibitory peptides can be disregarded.     

Broad characterization of endogenous peptides in the tree shrew visual system

Endogenous neuropeptides, acting as neurotransmitters or hormones in the brain, carry out important functions including neural plasticity, metabolism and angiogenesis. Previous neuropeptide studies have focused on peptide-rich brain regions such as the striatum or hypothalamus. Here we present an investigation of peptides in the visual system, composed of brain regions that are generally less rich in peptides, with the aim of providing the first broad overview of peptides involved in mammalian visual functions. We target three important parts of the visual system: the primary visual cortex (V1), lateral geniculate nucleus (LGN) and superior colliculus (SC). Our study is performed in the tree shrew, a close relative of primates. Using a combination of data dependent acquisition and targeted LC-MS/MS based neuropeptidomics; we identified a total of 52 peptides from the tree shrew visual system. A total of 26 peptides, for example GAV and neuropeptide K were identified in the visual system for the first time. Out of the total 52 peptides, 27 peptides with high signal-to-noise-ratio (>10) in extracted ion chromatograms (EIC) were subjected to label-free quantitation. We observed generally lower abundance of peptides in the LGN compared to V1 and SC. Consistently, a number of individual peptides showed high abundance in V1 (such as neuropeptide Y or somatostatin 28) and in SC (such as somatostatin 28 AA1-12). This study provides the first in-depth characterization of peptides in the mammalian visual system. These findings now permit the investigation of neuropeptide-regulated mechanisms of visual perception.     

Neuropeptide Y and somatostatin immunoreactive perikarya in preaortic ganglia projecting to the rat ovary

Postganglionic perikarya in preaortic ganglia projecting to the ovary of the rat were identified utilizing the retrograde fluorescent tracer, true blue. Ovarian perikarya were subsequently examined for neuropeptide Y and somatostatin immunoreactivity. True blue-labelled ovarian postganglionic perikarya were distributed in the coeliac ganglion and in smaller ganglia located at the origins of the renal and ovarian arteries. In the coeliac ganglia, 74.4 +/- 18.8% of true blue-labelled ovarian perikarya were immunoreactive to neuropeptide Y while 37.4 +/- 9.6% were immunoreactive to somatostatin. In the inferior smaller ganglia, 72.2 +/- 18.7% and 2.2 +/- 2.2% of the true blue-labelled ovarian perikarya were immunoreactive to neuropeptide Y and somatostatin respectively. It is suggested that neuropeptide Y and somatostatin participate in the modulation of ovarian function.     

Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

Cloned neuropeptide Y (NPY) Y1 and pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells show considerable agonist-driven internalization, in contrast to the NPY Y2 receptor.

Guinea-pig neuropeptide Y1 and rat pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y1 and Y2, but not the Y4, receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y2 receptor expressed in Chinese hamster ovary cells was small at 37 degrees C, and essentially absent at or below 15 degrees C, possibly in connection to the large molecular size of the receptor-ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 degrees C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y1 and the Y4 receptors could be blocked, and that of the Y2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y2 subtype. The restoration of Y1 and Y4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y1 and Y4 receptors have much larger subcellular dynamics than the Y2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes.     

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus)

Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.     

Immunohistochemical characteristics of nerve fibres supplying the porcine vas deferens. A colocalisation study

 Double-labelling immunofluorescence was used to investigate the coexistence of the catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase and several neuropeptides including neuropeptide Y, vasoactive intestinal polypeptide, Leu⁵-enkephalin, somatostatin, calcitonin gene-related peptide and substance P in nerve fibres supplying the vas deferens in juvenile and adult pigs. The study has revealed three major populations of nerve terminals innervating the organ: (1) noradrenergic fibres; (2) non-noradrenergic (putative cholinergic) fibres containing vasoactive intestinal polypeptide, neuropeptide Y and somatostatin, supplying almost exclusively the lamina propria; and (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. The population of noradrenergic nerves can be divided into three subpopulations: a somatostatin-containing, a Leu⁵-enkephalin-containing and a subpopulation immunonegative to the peptides investigated, in descending order of magnitude. Coexistence patterns of the substances existing within nerve fibres supplying the vas deferens blood vessels are clearly different from those found in nerve fibres innervating the organ wall. The majority of the noradrenergic fibres associated with blood vessels contain neuropeptide Y only, while non-noradrenergic perivascular nerves contain predominantly vasoactive intestinal polypeptide. The possibility of different sources of origin of the particular nerve fibre subpopulations supplying the porcine vas deferens and its blood vessels is discussed. Accepted: 23 October 1996     

Dexfenfluramine treatment and hypothalamic neuropeptides in diet-induced obesity in rats.

Neuropeptide Y (NPY) is a powerful appetite stimulant, and hypothalamic concentrations rise after food deprivation and in experimental diabetes. Serotonergic drugs such as dexfenfluramine are inhibitors of feeding. We measured hyothalamic NPY and NPY mRNA, along with galanin, neurotensin, and somatostatin in chow-fed rats and in rats with dietary obesity, and examined the effect of dexfenfluramine on these peptides in this model. Sixty-five rats were fed a palatable diet (condensed milk, sucrose and chow) for 6 weeks, which produced significant weight gain compared to twenty fed standard chow (145.1 +/- 2.3 g vs. 113.4 +/- 3.2 g, p less than 0.001). Groups of animals were treated for 7 days or 28 days with dexfenfluramine (1.8 mg/kg/day) or saline intraperitoneally via miniosmotic pumps. Hypothalami were dissected into medial and lateral blocks, and NPY, galanin, neurotensin, and somatostatin were measured by radioimmunoassay. Neuropeptide Y mRNA was measured by Northern blotting. Hypothalamic NPY was significantly higher in the palatable diet group compared to chow-fed controls (medial hypothalamus: 86.6 +/- 7.6 vs. 65.7 +/- 4.0 pmol/g tissue, p less than 0.02, lateral hypothalamus 71.2 +/- 6.6 vs. 53.1 +/- 3.6 pmol/g tissue, p less than 0.05), but NPY mRNA was unchanged. Although dexfenfluramine was effective at reducing weight gain in the animals fed the palatable diet, this did not result in any changes in the hypothalamic neuropeptides measured. Neuropeptide Y may be of importance in diet-induced obesity but the weight loss produced by dexfenfluramine in such animals is not mediated by changes in hypothalamic NPY.     

Neuroendocrine peptide levels in the gastrointestinal tract of mice after unilateral cervical vagotomy

The effects of left and right unilateral cervical vagotomy on the content of several neuroendocrine peptides were studied in different parts of the murine gastrointestinal tract, known to receive vagal innervation. The neuroendocrine peptides investigated were secretin, gastric inhibitory peptide (GIP), gastrin, motilin, peptide YY (PYY), somatostatin, substance P, VIP, neurotensin, neuropeptide Y (NPY), and galanin. The neuroendocrine peptide concentration was affected after both left and right vagotomy, and that the changes in the concentrations of the neuroendocrine peptide levels occurred in all the gastrointestinal segments investigated, namely antrum, small and large intestine. However, these changes varied, depending on which side was vagotomized and the interval after vagotomy. It is concluded that the vagus nerve had an important impact on the neuroendocrine system in the murine gut. It is suggested, furthermore that the contradictory results obtained earlier on the effect of vagotomy on the gastrointestinal peptides may depend on differences in the vagotomy methods used and on differences in observation time after vagotomy.     

Different long-term effects of bilateral and unilateral nucleus basalis lesions on rat cerebral cortical neurotransmitter content

Young adult rats received either unilateral or bilateral ibotenic acid infusions in their nucleus basalis, destroying most of the cholinesterase-staining neurons in that region. Cerebral cortex levels of choline acetyltransferase, somatostatin, neuropeptide Y, and monoamines were then assayed 2.5 and 10 months after bilateral lesions, or, 2.5, 10, and 14 months after unilateral lesions. Entorhinal and cerebral cortex levels of several amino acid transmitters were also measured. As expected, choline acetyltransferase activity was decreased in the frontal cortex ipsilateral to the ibotenic acid infusion in unilaterally or bilaterally lesioned animals. Parietal cortex concentrations of somatostatin and neuropeptide Y were altered by lesioning in a complicated, time-dependent manner. Thus, while unilateral lesions transiently decreased or had no effect on these neuropeptide levels, bilateral lesions elevated the level of each neuropeptide by over 100% at 10 months. Other cortical transmitter systems investigated appeared to be less affected by nucleus basalis-lesions. Unilateral lesions had no effect on prefrontal cortex norepinephrine, serotonin, or dopamine content at 14 months post-lesioning. These different neurochemical effects of unilateral and bilateral nucleus basalis lesions may be important for developing a model for the trans-synaptic effects of cortical cholinergic deafferentation.