A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues

Antisera towards neurotensin (NT) and the structurally related peptide, LANT6, were used to characterize immunoreactive peptides and proteins in extracts of chicken tissues. A 17 kDa protein was identified by Western blotting as a potential precursor to NT and LANT6. However, the posttranslational processing of this common precursor appeared to be tissue specific, giving rise to disproportionate amounts of NT and LANT6, along with varying expression of a large molecular LANT6 (M_r, 15 kDa). The intestinal cells containing immunoreactive NT, LANT6, and large molecular LANT6 behaved similarly during fractionation by size and density. These activities also banded together in particles resembling vesicles during centrifugation of isotonic homogenates of tissue. These results suggest that chicken NT and LANT6 are biosynthesized as parts of the same precursor, the processing of which can give rise to a variety of products stored within secretory vesicles.     

Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

Inhibition of neurotensin release by a cyclic hexapeptide analog of somatostatin

Studies were performed to determine whether the cyclic hexapeptide analog of somatostatin, cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe) II, could alter circulating levels of neurotensin (NT) and inhibit the release of NT from small intestine following the intraluminal perfusion of lipid and ETOH. The small intestine of anesthetized rats was perfused with 0.9% NaCl, 1mM ETOH, 100 mM ETOH or 1 mM oleic acid with and without the intravenous infusion of the somatostatin analog. Plasma samples collected from the superior mesenteric vein were extracted, chromatographed on HPLC and assayed with both C-terminal and N-terminal antisera to NT. The basal circulating levels of chromatographically and immunochemically identified NT observed during the perfusion of the small intestine with 0.9% NaCl were significantly lower (p less than 0.01) during the IV infusion of the somatostatin analog as compared to animals infused IV with saline. The 2-3 fold increase in plasma levels of NT observed with the intestinal perfusion of oleic acid and ETOH did not occur in animals simultaneously infused IV with the somatostatin analog. The somatostatin analog was also effective in decreasing the basal levels of NT metabolite NT(1-8) as well as inhibiting the increase in this metabolite that accompanies the stimulated release of NT.     

Isolation,structure and biologic activity of chicken intestinal neurotensin

Using a radioimmunoassay towards bovine neurotensin (NT), chicken NT has been purified to homogeneity from extracts of intestine and its amino acid sequence determined to be: <Glu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu-OH. The molecule is identical to the bovine peptide except for the 3 amino acid substitutions located in its NH₂-terminal half and italicized above (His/Tyr; Val/Glu; Ala/Pro). The structure for chicken NT is consistent with earlier immunochemical studies which indicated a COOH-terminal homology with bovine NT [1]. The peptide isolated was shown to be near equipotent with bovine NT in its ability to induce hypotension, hyperglycemia, and cyanosis in the anesthesized rat, underscoring the importance of the COOH-terminal residues in NT for biological activity.     

Neuropeptides and thermoregulation: the interactions of bombesin, neurotensin, TRH, somatostatin, naloxone and prostaglandins

In this study we have examined the interactions of bombesin (1 microgram ICV), neurotensin (1 microgram ICV), TRH (10 micrograms ICV), somatostatin (10 micrograms ICV), PGE2 (10 micrograms ICV) and naloxone (10 mg/kg SC) on thermoregulation in the rat at room temperature (20 +/- 1 degree C). Given alone, bombesin, neurotensin, somatostatin and naloxone all produced hypothermia (bombesin greater than neurotensin greater than somatostatin congruent to naloxone). PGE2 was hyperthermic, and TRH had no effect. Bombesin and PGE2 neutralized one another's effects. Neurotensin had no effect on PGE2-induced hyperthermia. Naloxone enhanced the hypothermic effect of bombesin and somatostatin enhanced the rate of onset of hypothermia after bombesin. TRH had no effect on bombesin-induced hypothermia. TRH, somatostatin and naloxone had no effect on neurotensin-induced hypothermia. TRH antagonized the hypothermia due to naloxone and somatostatin.     

生长抑素前体蛋白1和前脑啡肽原mRNA在鸡肠Remak神经中的分布

肠Remak神经(Intestinal nerve of Remak,INR)是禽类特有的一根自主神经节链,但INR中肽类递质的分布至今仍然存在许多疑问。本文应用RT-PCR方法从鸡脑组织提取的RNA中扩增生长抑素前体蛋白1(Somatostatin precursor1,PSS1)和前脑啡肽原(Preproenkephalin,PPE)基因片段,将其连接于pGM-Teasy质粒,经过转化大肠杆菌、挑取阳性克隆和测序鉴定,确定为目的片段。分别以线性化的SS1/pGM-Teasy和PPE/pGM-Teasy质粒为模板,用体外转录的方法合成正反义地高辛标记RNA探针。通过原位杂交方法将合成的探针用于探查PPE和PSS1 mRNA在鸡空回肠段和直肠段INR中的分布情况。结果表明:INR中大部分神经细胞中有PPE和PSS1的mRNA的转录,其中PPE探针杂交阳性细胞在空回肠段和直肠段INR分别占83.79%±7.96%和96.04%±4.53%,而PSS1探针在空回肠段和直肠段INR中的杂交阳性细胞分别占86.98%±7.93%和86.07%±6.11%;在整个INR中都可能有PPE和PSS1 mRNA共存于同一神经细胞的现象;原位杂交阳性神经细胞胞体呈有突起的梭形或椭圆形,其纵轴与INR延伸的方向平行;阳性神经细胞胞体在INR神经节中呈层状或成群分布,在节间束也有少量的阳性细胞分布。本文从基因水平证明INR中大量神经细胞进行PPE或PSS1的mRNA的转录,并可能作为外源性生长抑素1和脑啡肽能神经纤维支配到肠壁和输卵管     

Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation

Biochemical, histochemical and neurophysiological data suggest that substance P and somatostatin are neurotransmitters for primary afferent neurons. This study used intrathecal administration of these peptides and others (neurotensin and vasoactive intestinal polypeptide) in chronically catheterized, environmentally adapted, freely moving rats to evaluate their effects on unconditioned behavior. Substance P and somatostatin each elicited behaviors which were dose related. The behaviors included caudally directed biting and licking along with hindlimb scratching, writhing and retching. The behavioral responses were rapid in onset (1 min) and, in the case of substance P, short in duration (3 min). Vehicle, neurotensin and vasoactive intestinal polypeptide were without effect. These results demonstrate the ability of substance P and somatostatin to induce behavior in rats upon intrathecal administration and extend previous studies in mice.     

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Immunohistochemical identification of gastrointestinal neurotensin cells in human embryos

The endocrine gastro-entero-pancreatic (GEP) system of 10 human embryos was studied with special reference to neurotensin-immunoreactive cells. These cells are first present in the ileal and jejunal mucosa of 12 to 13 week old embryos. Thereafter the neurotensin-immunoreactive cells are found regularly in these segments of the gut with an increasing number towards the terminal ileum. At about the twentieth week of gestation, the neurotensin cells are detected also in the lower duodenum, i.e. the distribution pattern is more extensive in this age than in younger embryos or in adults.     

Neuronal systems immunologically related to the somatostatin system in the garden dormouse

Cells and fibers containing somatostain (SRIF) or SRIF-like peptides were detected immunocytochemically in the brain of the garden dormouse (Eliomys quercinus L.). The periventricular preoptic nucleus and the paraventricular nucleus encompass a component of the SRIF-immunoreactive hypothalamo-infundibular and hypothalamo-neurohypophyseal systems. The suprachiastmatic, ventromedial and arcuate nuclei contain a number of SRIF-positive cells and receive a rich SRIF innervation. The extrahypothalamic systems containing SRIF can be subdivided into the following groups: (1) Afferents, the cellular origin of which is not always clearly evident, i.e., fibers of the septum, the lateral preoptic area, the thalamus, the superior olivary nucleus, the mesencephalic gray matter, and the subfornical organ; (2) dispersed cells with short projections (neocortex, caudate nucleus, putamen); (3) scattered cells with short projections (nucleus accumbens) or innervating remote territories (nucleaus interpeduncularis); (4) vascular organ of the lamina terminalis, a neurohemal area comparable to the median eminence. These observations lead to a theory of a functional bipotentiality of the somatostatin molecule. Immunocytological results depend on the antisera employed, the type of fixation and the experimental conditions. Adrenalectomy is followed by an accumulation of immunoreactive material in all SRIF-containing systems of the brain. These results clearly indicate the SRIF participates in the function of the CRF-ACTH-adrenal axis. The endocrine disturbance induced by adrenalectomy appears to modify the activity of both the neurohormonal and neuromodulator components of the SRIF system.     

Histamine release by neurotensin in the rat hindquarter: structure-activity studies

Histamine releasing effects of neurotensin (NT) and several NT fragments and structural analogues were measured in the rat perfused hindquarter. The results show that the chemical groups responsible for histamine release are located in the C-terminal sequence Arg9-Pro10-Tyr11-Ile12-Leu13-OH. Both the spatial configuration and positive charge of Arg8 and Arg9 appear to contribute to the histamine releasing effect of NT. Optimization of the histamine releasing effect of NT requires both a free C-terminal carboxyl group and the presence in position 11 of NT of an aromatic residue, with the L-configuration, bearing an heteroatom capable of hydrogen bonding with the receptor. The results indicate that the structural requirements of NT to induce histamine release from the rat perfused hindquarter are similar to those involved in other peripheral biological actions of NT.     

A tetradecapeptide somatostatin dicarba-analog: Synthesis,structural impact and biological activity

We described here the first tetradecapeptide somatostatin-analogue where the disulfide bridge has been replaced by a carbon–carbon double bond. This analogue was prepared using microwave assisted ring closing metathesis (RCM) using the 2nd generation Grubbs as catalyst. Under our optimized conditions the cyclization between allylGly 3 and 14 proceeded in moderate yield, excellent cyclic/linear ratio and very high Z-double bond selectivity. NMR studies also demonstrated that the conformational flexibility of this peptide is increased in comparison to that of the natural hormone. Remarkably, this alkene-bridged somatostatin analog is highly selective against somatostatin receptors 1 and 5, suggesting that conformational rigidity is not required for the efficient interaction of somatostatin analogues with these two receptors.     

Distribution of CGRP-, somatostatin-, galanin-, VIP-, and substance P-immunoreactive nerve fibers in the chicken carotid body

Summary An immunoperoxidase method was used to investigate and compare the distribution of neuropeptide-immunoreactive (ir) nerve fibers and neurofilament-ir fibers in chick carotid body. The vagus nerve and its branches were intensely immunoreactive with an antiserum against chick neurofilaments. The branches from the vagus and the recurrent laryngeal nerves anastomosed within the connective tissue encircling the carotid body, and then entered the organ to form a network of neurofilament-ir fibers. Immunoreactivities for CGRP, somatostatin, galanin, VIP and substance P were found in the carotid body; they were located within varicose fibers. Immunoreactivity for each peptide was discretely and characteristically distributed. Dense networks of varicose CGRP-ir nerve fibers were found throughout the carotid body in close proximity to clusters of carotid body cells and to blood vessels. Substance P-ir fibers were distributed similarly to CGRP-ir fibers. Somatostatin-ir fibers appeared as patches distributed around chief cells. Numerous galanin- and VIP-ir nerve fibers were observed in the connective tissue surrounding the carotid body, but they occurred in only moderate densities in the parenchyma.     

Immunoreactive substance P in the chicken gut: Distribution,development and possible functional significance

Summary The distribution and cellular localization of substance P in the chicken gut was studied by immunocytochemistry and immunochemistry. Substance P-containing nerve fibers are numerous in the gut wall. They occur in the smooth muscle layer as well as in the mucosa, where they are associated with blood vessels or surround the intestinal crypts. The fibers are particularly numerous in the myenteric and submucosal plexuses, where substance P-containing nerve-cell perikarya are also encountered. Substance P was found also in scattered endocrine cells of the small intestine, caeca and colon. Previously, bombesin-containing cells, which are numerous in the proventriculus, have been mistakenly identified as substance P cells due to crossreactivity of certain antisera against substance P. Immunochemistry revealed the highest concentration of substance P in the duodenum. The gel chromatographic behavior of chicken substance P differs slightly from that of synthetic bovine substance P, suggesting that chicken substance P differs structurally from mammalian substance P. Substance P-containing nerve fibers in the chicken gut develop slowly after hatching, apparently beginning in the duodenum; at approximately 20 weeks after hatching the distribution pattern is fully developed.A functional investigation was performed on the isolated chicken caecum to clarify the role of substance P in the contractile behavior of smooth muscle. Substance P contracted the caecum over a wide dose range; the contractile response was greater in 20 week-old chickens than in 4 and 10 week-old animals. Electrical field stimulation caused a relaxation of the caecum and a contraction upon cessation of stimulation. Neither of these responses, both of which are neurally mediated, were inhibited by adrenergic and cholinergic blockade. It is conceivable that the contractile response following electrical stimulation is caused by substance P released from nerve fibers in the smooth muscle.     

Effects of somatostatin and a somatostatin agonist on diet-induced pancreatitis in mice

We examined the effects of somatostatin-14 and the potent somatostatin agonist (N-acetyl-[Des(Ala¹, Gly²),p-Cl-Phe⁶,D-Trp⁸]-somatostatin amide) on choline deficient, ethionine enrichèd diet (CDED)-induced acute pancreatitis in mice. Serum amylase determinations were performed, and specimens from the pancreas were examined by light and electron microscopy. No significant beneficial effects of somatostatin or its agonist were found in this model of acute pancreatitis.     

An immunohistochemical study of somatostatin in the stomach and the small intestine of the African ostrich (Struthio camelus)

The aim of the present study was to clarify the distribution and relative frequencies of somatostatin (SST)-producing cells in the stomach and the small intestine of the ostrich by using immunohistochemistry. The results indicated that somatostatin-immunoreactive (SST-IR) cells were distributed in mucosal layers of the proventriculus, duodenum, jejunum and ileum. However, no immunoreactivity was observed in the gizzard. SST-IR cells were found at the lower part of glandular lobule in the proventriculus, which were oval and round generally. SST-IR cells were present in the mucous membrane of entire small intestine of the ostrich. SST-IR cells had round and spherical shapes (closed-type cells), or spindle and pyriform shapes (open-type cells) in the small intestine. SST-positive cells were localized preferentially in the proventriculus of the 60-day-old ostrich. These results indicated that SST might be involved in functional and developmental regulation of gastrointestinal tract of the ostrich.     

Involvement of dopamine in the central effect of neurotensin on intestinal motility in rats

The effects of intracerebroventricular (ICV) administration of neurotensin (NT) before a meal on intestinal postprandial motility were examined in conscious rats chronically fitted with intraparietal Nichrome electrodes in the duodeno-jejunum. The effects were compared with those of two analogues, [D-Tyr¹¹]NT and [D-Trp¹¹]NT, resistant to degradation by brain peptidases. NT (10 μg ICV) delayed the occurrence of postprandial disruption of duodenal motility and blocked it on the jejunum. [D-Tyr¹¹]NT and [D-Trp¹¹]NT (1 μg ICV) elicited the same effects but at a ten-fold lower dose. NT administered peripherally just before a meal significantly lengthened the duration of the postprandial motor pattern. The central effect of NT on the fed pattern involved dopaminergic neurons as it was mimicked by dopamine, blocked by haloperidol and partly antagonized by either sulpiride or (+) SCH 23390. It is concluded that: 1) both D₁ and D₂ receptors are involved in the blocking effect of the postprandial disruption induced by central NT; 2) that [D-Tyr¹¹]NT and [D-Trp¹¹]NT are potent agonists at NT receptors in the brain.     

The stability and metabolism of intravenously administered neurotensin in the rat

The clearance and metabolism of synthetic and tritiated (³H) neurotensin (NT) were studied following its intravenous injection in a pharmacologic dose (500 pmol/kg) into anesthesized rats. Immunoreactive NT (iNT), measured in a radioimmunoassay (RIA) with use of a carboxyl-(C)-terminal directed antiserum, displayed an apparent half-life ($\text{t}\text{1}\text{2}$) of 0.55 min, while that measured by an amino-(N)-terminal directed antiserum had a $\text{t}\text{1}\text{2}$ of 5 min. The radiolabel from injected ³H-NT (³H on Tyr^3,11) had a $\text{t}\text{1}\text{2}$ of 6.5 min. High-pressure liquid chromatography of extracts of plasma obtained from the circulation 0.5–3 min after injection of NT and ³H-NT showed the presence of NT and the generation mainly of the fragments NT^1–8, NT^1–11, and NT^9–13, as well as free ³H-labeled tyrosine. The apparent half-lives of intravenously injected synthetic NT^1–8, NT^1–11 and NT^1–12 measured with the N-terminal RIA were 9, 5 and 5 min, respectively, while that for NT^9–13 was less than 0.5 min. These results indicate that exogenously injected NT is rapidly metabolized to form N-terminal fragments which are cleared more slowly than NT. These findings suggest that use of N-terminal antisera to detect the release of endogenous NT into the circulation is likely to yield measurements of the fragments NT^1–8 and NT^1–11 which thus far have been found to be biologically inactive.