Degradation of vasoactive intestinal polypeptide by tissue homogenates

Extracts of liver, kidney and brain contain an enzyme that is highly specific for degradation of vasoactive intestinal polypeptide (VIP). The Michaelis constants (K_m's) appear to be nearly identical in all three tissues, averaging about 10^?5 mol/liter. The V_max for kidney and liver are about the same but that for cerebral cortex is about two-fold lower. Since the relative V_max in the three organs differ for insulin and VIP, it is concluded that it is unlikely that the same enzyme is responsible for the degradation of both peptides.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

Dual regulation of ACTH secretion by guanine nucleotides in permeabilized AtT-20 cells

1. We have examined the effects of guanine nucleotides on ACTH secretion from digitonin-permeabilized AtT-20 cells, with the aim of analyzing the involvement of GTP-binding proteins (G proteins) in the secretory process. 2. AtT-20 cells permeabilized with 20 microM digitonin displayed calcium-dependent secretion. The EC50 of calcium was approximately 2 microM and the maximal stimulation was 350% of basal release. 3. Nonhydrolyzable guanine nucleotides also stimulated ACTH release, in a virtually Ca2+-free medium. The EC50 of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) was approximately 15 microM and the maximal stimulation was approximately 230% of basal release. The effects of calcium and guanine nucleotides were not additive. 4. In the presence of the inhibitory hormone, somatostatin guanine nucleotides inhibited the calcium-stimulated secretion. 5. Both the stimulatory and the inhibitory effects on secretion of guanine nucleotides were independent of changes in cyclic AMP (cAMP) and calcium. It is suggested that G proteins influence an unknown step in the secretion process, which would be near or at the exocytotic site. 6. The results can be explained by assuming the existence of two types of G proteins, one with stimulatory effects on exocytotic release (GeS) and another with inhibitory effects (GeI).     

Influence of pregnancy and sex steroids on concentration, motor effect and receptor binding of VIP in the rabbit female genital tract

The influence of sex steroid and pregnancy on the tissue concentration, uterine motor effect and receptor binding of VIP has been studied in the female genital tract of pregnant rabbits and oophorectomized rabbits during progesterone and/or oestrogen substitution. The concentration of immunoreactive VIP was high in the vagina and cervix, and lower in the uterine body of both pregnant and non-pregnant rabbits. A significant decrease in the VIP concentration (pmol/g wet weight) of the uterine body was observed toward term of pregnancy. The total uterine content of VIP, however, seems unchanged. Treatment of oophorectomized rabbits with ovarian steroids had no effect on the VIP concentration. The sensitivity for and potency of VIP on the relaxation of uterine muscle was significantly higher in oophorectomized rabbits treated with a combination of progesterone and oestrogen than in control rabbits. No difference was observed between non-pregnant and pregnant rabbits. The degradation and binding affinity for 125I-labelled VIP was highest in oophorectomized rabbits substituted with both oestrogen and progesterone. In the pregnant rabbits, the amount of receptors was decreased near term. In conclusion, sex steroids are able to influence the motor effect of VIP at the receptor level, but have no effect on the VIP concentration in the female genital tract.     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.     

Effects of PHI on vasoactive intestinal peptide receptors and adenylate cyclase activity in lung membranes. A comparison in man, rat, mouse and guinea pig

The presence of receptors, recognized by vasoactive intestinal peptide (VIP) as well as by PHI (a peptide with N-terminal histidine and C-terminal isoleucine amide), was documented in lung membranes from rat, mouse, guinea pig and man by the ability of these receptors, once occupied, to stimulate adenylate cyclase. In lung membranes from rat, mouse and guinea pig, the capacity of VIP, PHI and secretin to stimulate the enzyme and the potency of the same peptides to compete with 125I-VIP for binding to VIP receptors were similar, the affinity decreasing in the order: VIP greater than PHI greater than secretin. In addition, dose-effect curves were compatible with the coexistence of high-affinity and low-affinity VIP receptors, in the four animal species considered. If PHI was able to recognize all VIP receptors it could not, however, discriminate the subclasses of VIP receptors.     

Distribution and vasomotor effects of peptide HI (PHI) in feline cerebral blood vessels in vitro and in situ

Peptide HI (PHI)-immunoreactive nerve fibres were numerous around cerebral blood vessels of the cat. The number and distribution resemble that previously found for vasoactive intestinal polypeptide (VIP), a peptide with which PHI co-exists in pial arteries, at least in some segments. PHI and VIP elicit dilatation in a concentration-dependent manner in isolated middle cerebral arteries; the maximum effects were similar but VIP was considerably more potent. Neither effect was blocked by atropine, cimetidine or propranolol, confirming an action at a non-adrenergic, non-cholinergic site. In chloralose-anaesthetized cats PHI and VIP elicited concentration-dependent dilatations; the magnitude of responses was similar, however, considerably more PHI was necessary to elicit the same response as that of VIP. The results suggest that though both peptides are co-localized and may act at the same receptor, VIP is a more likely candidate for eliciting dilatation during physiological conditions.     

Interaction of secretin5–27 and its analogues with hormone receptors on pancreatic acini

The C-terminal tricosapeptide of secretin (S_5–27) and two analogues, one with asparagine replacing aspartic acid in position 15 (15-Asn-S_5–27) and one with lysine replacing aspartic acid in position 15 (15-Lys-S_5–27) were tested for their abilities to interact with hormone receptors on pancreatic acinar cells. In interacting with the receptors which prefer vasoactive intestinal peptide (vaso-active intestinal peptide-preferring receptors), the apparent affinity of 15-Asn-S_5–27 was equal to that of 15-Lys-S_5–27 and was greater than that of S_5–27. In interacting with secretin-preferring receptors, the apparent affinity of 15-Asn-S_5–27 was equal to that of S_5–27 and was greater than that of 15-Lys-S_5–27. In interacting with the secretin-preferring receptors each of the secretin fragments was approximately 2% as effective as secretin in causing an increase in cellular cyclic AMP. None of these fragments was able to cause a detectable increase in cyclic AMP mediated by the vasoactive intestinal peptide-preferring receptors. The dose vs. response curves for the action of secretin and vasoactive intestinal peptide on cellular cyclic AMP and on amylase secretion as well as the patetern of effects of secretin fragments on these actions indicated that the increase in amylase secretion caused by vasoactive intestinal peptide and secretin is mediated exclusively by the vasoactive intestinal peptide-preferring receptors. Furthermore, occupation of approximately 50% of the vasoactive intestinal peptide-preferring receptors is sufficient to cause maximal stimulation of amylase secretion.     

The effect of VIP on guinea pig taenia coli requires the whole sequence

Truncated sequences of VIP_1–28 i.e., VIP_1–6, VIP_15–28 and VIP_18–28, were synthesized. The biological activity of the peptides was tested on the isolated taenia coli from guinea-pig. Unlike VIP_1–28, the truncated peptides had no effect alone or in combination. We also synthesized two peptides where VIP_1–6 or VIP_1–9 were joined with VIP_20–28 or VIP_21–28, respectively, with omission of the mid portion of VIP_1–28. These peptides had no detectable biological activity. Finally, we synthesized Gly^17,18,19-VIP, and tested it in the above mentioned system. It had a greatly reduced bioactivity compared with native VIP.     

The dependence on gp130 cytokines of axotomy induced neuropeptide expression in adult sympathetic neurons

Adult peripheral neurons exhibit dramatic changes in gene expression after axonal injury, including changes in neuropeptide phenotype. For example, sympathetic neurons in the superior cervical ganglion (SCG) begin to express vasoactive intestinal peptide (VIP), galanin, pituitary adenylate cyclase activating polypeptide (PACAP), and cholecystokinin after axotomy. Before these changes, nonneuronal cells in the SCG begin to express leukemia inhibitory factor (LIF). When the effects of axotomy were compared in LIF?/? and wild‐type mice, the increases in VIP and galanin expression were less in the former, though significant increases still occurred. LIF belongs to a family of cytokines with overlapping physiological effects and multimeric receptors containing the subunit gp130. Real‐time PCR revealed large increases in the SCG after axotomy in mRNA for three members of this cytokine family, interleukin (IL)‐6, IL‐11, and LIF, with modest increases in oncostatin M, no changes in ciliary neurotrophic factor, and decreases in cardiotrophin‐1. To explore the role of these cytokines, animals with selective elimination of the gp130 receptor in noradrenergic neurons were studied. No significant changes in mRNA levels for VIP, galanin, and PACAP were seen in axotomized ganglia from these mutant mice, while the increase in cholecystokinin was as large as that seen in wild‐type mice. The data indicate that the inductions of VIP, galanin, and PACAP after axotomy are completely dependent on gp130 cytokines and that a second cytokine, in addition to LIF, is involved. The increase in cholecystokinin after axotomy, however, does not require the action of these cytokines. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

神经肽Y(NPY)的生理功能研究进展

神经肽Y(NPY)是机体内的一种重要且保守的神经递质,一般以前体形式存在,释放的有活性的NPY主要通过与其受体结合发挥作用。NPY受体包含了亚型Y1、Y2、Y3、Y4、Y5、Y6、Y7、Y8。Y1和Y2是NPY发挥收缩血管作用的关键受体;Y1、Y2和Y5是NPY调节动物摄食行为的关键受体;Y1、Y2和Y4是NPY调控动物焦虑、沮丧行为的必要受体。着重对NPY与其各种受体结合后如何行使动物的相关生理功能的情况进行了阐述。     

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.     

Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors inDictyostelium discoideum

Summary Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolatedDictyostelium discoideum membranes. The K_0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 x 10⁻⁴, 1.3 X 10⁻², and higher than 0.1 s⁻¹. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (K_D is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal inD. discoideum.     

Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex

The Na+-motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated delta psi (inside positive) and delta pH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na+ in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an electrochemical potential of Na+ by the influx of Na+.     

Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization

Activated human neuropeptide Y Y(1) receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y(2) receptors that neither internalize nor desensitize. To identify motifs implicated in Y(1) receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y(2) C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [phi-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for beta-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y(1) receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and beta-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of beta-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y(1) receptors nor beta-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y(1) desensitization. In the Y(2) receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, beta-arrestin activation, internalization and recycling. Overall, our results indicate that beta-arrestin-mediated desensitization and internalization of Y(1) and Y(2) receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.     

Vasoactive intestinal polypeptide-like immunoreactivity in nerves of the pancreatic islet of the teleost fish,Gillichthys mirabilis

Summary In the teleost fish, Gillichthys mirabilis, vasoactive intestinal polypeptide (VIP)-like immunoreactivity is present in some nerve fibres of the principal pancreatic islet and surrounding tissues, the vagus and splanchnic nerves, the coeliac ganglion and the wall of the intestine. The nerves of the pancreatic islet that contain VIP-like immunoreactivity probably correspond to one of the two types of non-cholinergic, non-adrenergic (peptidergic) nerve previously described in this fish. Similarities in the localisation of hormonal peptides in fish and mammals suggest that the regulation of gastroenteropancreatic physiology in fish may resemble that of mammals.This work was supported by grants from the Medical Research Council of Great Britain and the National Institutes of Health, USA (Grant No. AM 17161)     

Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Binding of palladium(II) complexes to guanine, guanosine or guanosine 5-monophosphate in aqueous solution: potentiometric and NMR studies

The interaction of guanine, guanosine or 5^′-GMP (guanosine 5^′-monophosphate) with [Pd(en)(H₂O)₂](NO₃)₂ and [Pd(dapol)(H₂O)₂](NO₃)₂, where en is ethylenediamine and dapol is 2-hydroxy-1,3-propanediamine, were studied by UV-Vis, pH titration and ¹H NMR. The pH titration data show that both N1 and N7 can coordinate to [Pd(en)(H₂O)₂]²⁺ or [Pd(dapol)(H₂O)₂]²⁺. The pK_a of N1-H decreased to 3.7 upon coordination in guanosine and 5^′-GMP complexes, which is significantly lower than that of ∼9.3 in the free ligand. In strongly acidic solution where N1-H is still protonated, only N7 coordinates to the metal ion, but as the pH increases to pH ∼3, ¹H NMR shows that both N7-only and N1-only coordinated species exist. At pH 4-5, both N1-only and N1,N7-bridged coordination to Pd(II) complexes are found for guanosine and 5^′-GMP. The latter form cyclic tetrameric complexes, [Pd(diamine)(μ-N1,N7-Guo]₄⁴⁺ and [Pd(diamine)(μ-N1,N7-5^′-GMP)]₄H_x^(4−x)−, (x=2,1, or 0) with either [Pd(en)(H₂O)₂](NO₃)₂ or [Pd(dapol)(H₂O)₂](NO₃)₂. The pH titration data and ¹H NMR data agree well with the exception that the species distribution diagrams show the initial formation of the N1-only and N1,N7-bridged complexes to occur at somewhat higher pH than do the NMR data. This is due to a concentration difference in the two sets of data.     

Evidence against cyclic GMP as a mediator of the actions of secretagogues on amylase release from guinea-pig pancreas

In dispersed acini from guinea-pig pancrease several pancreatic secretagogues increased calcium outflux, cyclic GMP and amylase secretion, whereas nitroprusside and hydroxylamide increased cyclic GMP but did not increase calcium outflux or amylase secretion and did not alter the action of secretagogues on calcium outflux or amylase secretion. Secretin and vasoactive intestinal peptide increased cyclic AMP and increased secretion but did not alter cyclic GMP. Nitroprusside and hydroxylamine did not alter cyclic AMP or the action of secretin or vasoactive intestinal peptide on cyclic AMP and enzyme secretion. Agents that increased cyclic GMP also caused release of the nucleotide into the extracellular medium; however, this release did not correlate with secretion of amylase into the extracellular medium. 8-Bromo cyclic AMP as well as 8-bromo cyclic GMP increased enzyme secretion and potentiated the increase in enzyme secretion caused by cholecystokinin or carbachol. The increase in amylase secretion caused by vasoactive intestinal peptide or secretin plus either of the cyclic nucleotide derivatives was the same as that caused by the peptide alone. These results indicate that cyclic GMP does not mediate the action of secretagogues on pancreatic enzyme secretion, that the release of cyclic GMP into the extracellular medium does not occur by exocytosis and that the increase in enzyme secretion caused by 8-bromo cyclic GMP results from its stability to mimic the action of endogenous cyclic AMP.