Interaction of PHM, PHI and 24-glutamine PHI with human VIP receptors from colonic epithelium: comparison with rat intestinal receptors

PHM, the human counterpart of porcine Peptide Histidine Isoleucine amide (PHI), is shown to be a VIP agonist with low potency on human VIP receptors located in colonic epithelial cell membranes. Its potency is identical to that of PHI but by 3 orders of magnitude lower than that of VIP itself in inhibiting 125I-VIP binding and in stimulating adenylate cyclase activity. This contrasts markedly with the behaviour of PHI on rat VIP receptors located in intestinal epithelial cell membranes where PHI is a potent agonist with a potency that is 1/5 that of VIP. In another connection, we show that 24-glutamine PHI has the same affinity as 24-glutamic acid PHI (the natural peptide) for rat or human VIP receptors. These results indicate that while PHI may exert some physiological function through its interaction with VIP receptors in rodents, its human counterpart PHM is a very poor agonist of VIP in human. Furthermore, they show that the drastic change in position 24 of PHI (neutral versus acid residue) does not affect the activity of PHI, at least on VIP receptors.     

Interaction of Gila monster venom with VIP receptors in intestinal epithelium of human. A comparison with rat

Gila monster venom (1-300 micrograms/ml) is shown to inhibit completely the binding of [125I]VIP to human and rat intestinal epithelial cell membranes. In both models, the venom inhibits [125I]VIP binding and stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP and a potency that is 10000-50000 times lower than that of the peptide, on a weight basis. At maximal doses, VIP and Gila monster venom do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the venom occurs through VIP receptors. As is the case for VIP, adenylate cyclase activation by Gila monster venom requires the presence of GTP in the incubation medium. Finally, no VIP-like immunoreactivity was detected in the venom using an antiserum raised against mammalian VIP. All these data suggest the presence in the venom of the Gila monster, of a new substance which behaves as a VIP agonist in human as well as rat intestine.     

Effect of intestinal ischaemia on intestinal VIP levels and VIP interaction with intestinal epithelial cells from rat

1. The number (but not the affinity) of vasoactive intestinal peptide (VIP) receptors in small intestinal epithelial cells decreased following intestinal ischaemia in rats as compared to sham-operated animals. 2. There was a parallel decrease of the efficiency (but not the potency) of the neuropeptide upon cyclic AMP formation at the same level after intestinal ischaemia. 3. The surgical manipulation did not modify the level of VIP immunoreactivity in the gut segment studied. 4. These results suggest that the VIPergic system is not directly involved in the high loss of water and electrolytes that appears following intestinal ischaemia.     

Vasoactive intestinal peptide (VIP) binding to solubilized material from rat liver plasma membranes

A non-ionic detergent such as Lubrol-PX extracts in soluble form the VIP-binding structures of rat liver plasma membranes. Detergent-solubitized proteins bind specifically [¹²⁵I]VIP and the complex tracer-protein is identified by the use of Sepharose 6B columns. The interaction is only possible in the absence of detergent (below 0.001%) and is inhibited by native peptide. A molecular weight of about 80,000 was estimated for VIP-binding proteins by reference to a series of globular markers of proteins. Binding to VIP soluble proteins is specific and dependent on time as studied by the Hummel and Dreyer (Biochim. Biophys. Acta 63:530–532, 1962) assay.     

Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver

Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.     

Effect of gastroduodenostomy on intestinal vasoactive intestinal peptide (VIP) levels, and VIP binding and VIP stimulation of cyclic AMP in intestinal epithelial cells from rat

The concentration of VIP in duodenum and jejunum as well as the interaction of VIP (binding and stimulation of cyclic AMP accumulation) with epithelial cells from both gut segments were studied in rats after surgical bypass of the pylorus by gastroduodenostomy. Duodenal VIP concentration increased in rats with gastroduodenostomy as compared to sham-operated animals. The binding capacity (but not the affinity) of VIP binding sites and the efficiency (but not the potency) of VIP on cyclic AMP accumulation decreased in the condition of gastroduodenostomy. However, no modifications in either VIP concentration and interaction could be seen at the jejunal level.     

A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors

A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.     

Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages.

Functional vasoactive intestinal peptide (VIP) receptors have been characterized in rat peritoneal macrophages. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of two classes of binding sites: a class with high affinity (kd = 1.1 +/- 0.1 nM) and low capacity (11.1 +/- 1.5 fmol/10(6) cells), and a class with low affinity (kd = 71.6 +/- 10.2 nM) and high capacity (419.0 +/- 80.0 fmol/10(6) cells). Structural requirements of these receptors were studied with peptides structurally or not structurally related to VIP. Several peptides inhibited 125I-VIP binding to rat peritoneal macrophages with the following order of potency: VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, somatostatin, pancreastatin and octapeptide of cholecystokinin (CCK 26-33) were ineffective. VIP induced an increase of cyclic AMP production. Half-maximal stimulation (ED50) was observed at 1.2 +/- 0.5 nM VIP, and maximal stimulation (3-fold above basal levels) was obtained between 0.1-1 microM. Properties of these binding sites strongly support the concept that VIP could behave as regulatory peptide on the macrophage function.     

肠道上皮与肠道微生物相互作用的研究现状

肠道上皮是肠上皮细胞及其分泌物有机构成的黏膜界面。随着技术的进步和对肠道菌群作用的逐渐重视,研究者对肠道上皮与肠道微生物相互作用的认识也不断深入。研究表明,肠道上皮调节并维持肠道微生物的定殖与分布,肠道微生物也影响肠道上皮的多种屏障功能,二者通过一系列细胞分子机制紧密联系,共同维持肠道稳态。此外,其过程中产生的宿主-肠道菌群共代谢物被发现可以反映宿主的生理病理状态,作为指标被应用于临床疾病诊断、治疗效果评估和预后推测。本文基于近年的研究,综述了肠道上皮与肠道微生物的相互作用及其细胞分子机制,为进一步研究和临床应用总结了理论基础,并探讨了未来可能的研究方向。     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.     

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.     

Rapid formation of N-hydroxymethylpentamethylmelamine by mitochondria from rat small intestinal epithelium

Isolated rat intestinal mitochondria showed a considerable capacity to convert hexamethylmelamine to its monodemethylated metabolite pentamethylmelamine. Mitochondrial turnover rate is about the same as compared with microsomal preparations. Only in mitochondrial incubations N-hydroxymethylpentamethylmelamine could be identified as a metabolic intermediate. The known chemical reactivity of carbinolamines means that this activation pathway in mitochondria could be involved in the pharmacological or toxic effects of hexamethylmelamine.     

Binding and internalization of VIP in rat intestinal epithelial cells.

We have prepared villous cells from the jejunum of the rat small intestine and studied the effects of divalent cations and bacitracin on the binding and internalization of VIP. Villous epithelial cells (4 x 10(6) cells/ml) were suspended in a Hepes-NaCl buffer with 1.0% BSA, (pH 7.4) and the cells were incubated for varying periods of time with 125I-VIP at 24 degrees C. Specific binding of radiolabeled VIP was maximal within 10 min (10%) and slowly declined to 9.0 percent after 30 min. In the presence of 1.0 mg/ml bacitracin, however, maximal specific binding of VIP was only 2.7 percent (P less than or equal to 0.001). The addition of CA2+ or Mg2+ to the buffer significantly decreased binding of VIP in a concentration dependent manner. At 8.0, 4.0, 2.0 and 1.0 mM Ca2+, binding of 125I-VIP decreased by 70, 60, 40 and 25 percent, whereas in the presence of the same concentrations of Mg2+ binding was decreased to 50, 38, 25 and 10 percent (P less than or equal to 0.01). To determine if epithelial cells internalize VIP, we bound 125I-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150 micrograms/ml). Surface bound radioligand was the same at both 24 and 4 degrees C (5.3%), while internalized 125I-VIP was 4.0% at 24 degrees C compared to only 1.0% at 4 degrees C (P less than or equal to 0.001). At 24 and 4 degrees C, both Ca2+ (4.0 mM) and Mg2+ (8.0 mM) decreased surface bound radioligand by 60 percent (P less than or equal to 0.01) and lowered internalized radioactivity. These data demonstrate that (1) bacitracin decreases the binding of VIP to small intestinal epithelial cells, (2) both Ca2+ and Mg2+ affect the binding of VIP to its surface receptor and (3) VIP is internalized into epithelial cells.     

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

Effect of freezing on the coupling of VIP receptors to adenylate cyclase in rat liver membranes

In fresh rat liver plasma membranes, high affinity VIP receptors were specifically labelled with [125I] helodermin and were well coupled to adenylate cyclase while low affinity VIP receptors were not. After freezing and thawing low affinity VIP receptors were also coupled to adenylate cyclase. This modification of adenylate cyclase activation was specific for the VIP response as freezing and thawing did not modify Gpp (NH)p, NaF and glucagon stimulations.