Evidence for a C-terminal turn in PBAN An NMR and distance geometry study

The solution conformation of the pheromone biosynthesis activating neuropeptide (PBAN) of the moth Helicoverpa zea has been determined using homonuclear two-dimensional nuclear magnetic resonance techniques and distance geometry-restrained energy minimization. The insect peptide hormone showed a random distribution of conformers in aqueous solution, whereas in a less polar medium of trifluoroethanol and water, a reordering process was observed. In particular, the C-terminal region (Phe-Ser-Pro-Arg-Leu-NH₂) adopts a type I′β-turn conformation, residues 20-27 are in a helix conformation, and residues 1-19 exhibit a high degree of flexibility. Direct observation of the C-terminal β-turn configuration of PBAN is consistent with a previous report that showed a rigid, cyclic analog of the C-terminal pentapeptide of PBAN retained pheromonotropic activity [Nachman, R.J., Kuniyoshi, H., Roberts, V.A., Holman, G.M. & Suzuki, A. (1993). Biochem. Biophys. Res. Commun. 661-666], Furthermore, our results show no interaction between the C-terminal turn and the rest of the polypeptide chain, thus providing further evidence that the C-terminal turn is indeed the important conformation recognized by the PBAN receptor. © Munksgaard 1996.     

Addition of the (28–38) peptide sequence of PACAP to the VIP sequence modifies peptide selectivity and efficacy

Chimeric peptides were synthesized by adding the C-terminal extension 28-38 of the pituitary adenylate cyclase activating polypeptide (PACAP) to the sequences (1–27), (2–27), (3–27) and (6–27) of VIP. The capacity of these peptides to occupy the selective PACAP- and the non-selective PACAP-VIP receptors and to stimulate adenylate cyclase activity was studied in Chinese hamster ovary (CHO) cells expressing the recombinant receptors. The results were compared to those obtained with VIP and the corresponding VIP fragments. The presence of the (28–38) PACAP extension increased at least 100-fold the VIP- or VIP fragment affinities for the selective PACAP receptor but not for the non-selective PACAP-VIP receptors. Furthermore, on both receptors, the extension increased peptide intrinsic activity: VIP(3–28) was a partial agonist; while VIP(3–27)/PACAP(28–38) was as potent as VIP and was apparently a full agonist; VIP(6–28) had no intrinsic activity, but VIP(6–27)/PACAP(28–38) was a partial agonist. These results suggest: (1) the presence of a specific domain for the (28–38) PACAP sequence on the selective PACAP receptor; and (2) a stabilizing effect of the (28–38) PACAP sequence on the structure of N-terminally truncated VIP. © Munksgaard 1996.     

Regional haemodynamic responses to pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide in conscious rats.

1. Regional haemodynamic responses to the homologous peptides, pituitary adenylate cyclase-activating peptide (1-27) (PACAP27) and vasoactive intestinal polypeptide (VIP) were assessed by giving 20 min infusions (1.5-15 nmol kg-1 h-1) in conscious, chronically-instrumented, Long Evans rats. 2. PACAP27 caused dose-dependent depressor and tachycardic effects associated with renal, mesenteric and hindquarters vasodilatations, although only in the latter vascular bed was there a sustained increase in flow. 3. VIP caused dose-dependent depressor and tachycardic effects that were not significantly different from those caused by equimolar doses of PACAP27. However, the hindquarters vasodilator effects of VIP (at 7.5 and 15 nmol kg-1 h-1) were greater than those of PACAP27 (at the same doses), and accompanied by reductions in renal and mesenteric flows and conductances. 4. In the presence of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 11 mumol kg-1 h-1), there was significant inhibition of the hindquarters vasodilator effects of PACAP27 and VIP (at 7.5 and 15 nmol kg-1 h-1). Under these circumstances the renal and mesenteric vasoconstrictor effects of VIP were abolished. 5. The beta 2-adrenoceptor antagonist, ICI 118551 (670 nmol kg-1 bolus, 335 nmol kg-1 h-1 infusion), reduced the matched hindquarters vasodilator responses to PACAP27 (15 nmol kg-1 h-1) and VIP (7.5 nmol kg-1 h-1), and also abolished the renal vasoconstrictor effects of VIP. 6. The AT1-receptor antagonist, losartan potassium (20 mumol kg-1), had no significant effect on the haemodynamic response to PACAP27 (15 nmol kg-1 h-1), but augmented the hypotensive action of VIP (7.5 nmol kg-1 h-1). This influence of losartan was associated with conversion of the renal and mesenteric vasoconstrictor effect of VIP to vasodilatation. 7. Our findings show that similar changes in mean systemic arterial blood pressure in response to PACAP27 and VIP conceal substantial differences in their regional haemodynamic actions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264 , 82–85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C^α torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (α_L) and that in family 2 is right-handed helical (α_R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereo-chemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. © Munksgaard 1995.     

Interactions of VIP,secretin and PACAP(1-38) with phospholipids: a biological paradox revisited

Vasoactive intestinal peptide (VIP), secretin and pituitary adenylate cyclase-activating peptide(1-38)(PACAP(1-38)) are widely distributed amphipathic mammalian neuropeptides that exert diverse biological effects in target tissues located distant from their site of release. However, the half-life of exogenously-administered VIP, secretin and PACAP(1-38) in the bloodstream is relatively short (minutes) due to rapid degradation and inactivation. This seemingly paradoxical behavior suggests the presence of an innate system(s) that protects the peptides from degradation in vivo. To this end, VIP, secretin and PACAP(1-38) express distinct biophysical properties that once released may protect them from degradation in biological fluids. They self-aggregate at low (nanomolar) concentrations and interact avidly with biomimetic phospholipid monolayers and bilayers at physiological concentrations. The latter evokes conformational transition of the VIP, secretin and PACAP(1-38) molecules from predominantly random coil in aqueous solution to alpha-helix, the preferred peptide conformation for receptor interaction, in phospholipids. These features increase peptide stability and amplify bioactivity in vivo. Collectively, these data suggest the presence of an endogenous targeted delivery platform for VIP, secretin and PACAP(1-38). This innate system may constitute a novel molecular recognition paradigm that could also apply to other amphipathic neuropeptides. Importantly, the distinct behavior of VIP, secretin and PACAP(1-38) in the presence of phospholipids could be exploited to develop novel, long-acting therapeutic formulations of these peptides.     

Effect of vasoactive intestinal peptide (VIP)-related peptides on cholinergic neurogenic and direct mucus secretion in ferret trachea in vitro

1 We investigated whether vasoactive intestinal peptide (VIP) and its related peptides, pituitary adenylate cyclase activating peptide (PACAP) and secretin, regulate cholinergic neural mucus secretion in ferret trachea in vitro, using 35SO4 as a mucus marker. We also studied the interaction between VIP and secretin on cholinergic mucus output. 2 VIP (1 and 10 microM) increased secretion, whereas neither PACAP1 - 27, PACAP1 - 38 nor secretin (up to 10 microM) increased mucus output. In contrast, VIP, PACAP1 - 27 and PACAP1 - 38 concentration-dependently inhibited cholinergic neural secretion, with an order of potency of VIP>PACAP 1 - 38>PACAP1 - 27. Neither PACAP1 - 27 nor PACAP1 - 38 altered the secretion induced by acetylcholine (ACh). 3 Secretin increased cholinergic neural secretion with a maximal increase of 190% at 1 microM. This potentiation was blocked by VIP or atropine. Similarly, secretin (1 microM) potentiated VIP (1 microM)-induced mucus output by 160%. Secretin did not alter exogenous ACh-induced secretion. VIP vs secretin competition curves suggested these two peptides were competing reversibly for the same receptor. 4 We conclude that, in ferret trachea in vitro, VIP and PACAPs inhibit cholinergic neural secretion via pre-junctional modulation of cholinergic neurotransmission. VIP and secretin compete for the same receptor, possibly a VIP1 receptor, at which secretin may be a receptor antagonist.     

Comparative effects of PACAP and VIP on pancreatic endocrine secretions and vascular resistance in rat.

1. The effects of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and secretin on pancreatic endocrine secretions and vascular resistance were investigated and compared in the isolated perfused pancreas of the rat. The PACAP/VIP receptor types involved have been characterized. 2. On insulin secretion, in the range 10(-11) to 10(-8) M, PACAP and VIP elicited a concentration-dependent biphasic response from pancreas perfused with 8.3 mM glucose; the peptides were equipotent. In contrast, secretin was ineffective in the range 10(-11) to 10(-9) M; at 10(-8) and 10(-7) M, it induced only low and transient insulin responses. On the other hand, the peptides did not modify the basal insulin release in the presence of a non stimulating glucose concentration (2.8 mM). 3. On glucagon secretion, PACAP and VIP (10(-11) to 10(-8) M) but also secretin (10(-9) to 10(-7) M) caused a concentration-dependent peak shaped response from pancreas perfused with 2.8 mM glucose; PACAP and VIP were equipotent and 20 times more potent then secretin. On the other hand, the peptides did not affect the glucagon release in the presence of 8.3 mM glucose. 4. On pancreatic vessels, in the range 10(-11) to 10(-9) M, the three peptides were equipotent in inducing a concentration-dependent sustained increase in pancreatic flow rate. On the other hand, at the high concentration of 10(-7) M PACAP but not VIP provoked a transient decrease of flow rate. 5. This study provides evidence for PACAP/VIP type II receptors mediating insulin and glucagon secretion as well as vasodilatation in rat pancreas. In addition, the different efficacies of secretin suggest that these effects are mediated by different PACAP/VIP type II receptor subtypes.     

Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant

The vasoactivity of the 27- and 38-amino acid forms of the novel peptide pituitary adenylate cyclase-activating polypeptide (PACAP) was tested in vitro. Both forms of PACAP caused endothelium-independent vasodilation (assayed by their vasodilator action on rabbit aorta). When superfused for 1 min the relaxation EC50 of PACAP27 was 23 +/- 8 nM and of PACAP38 was 152 +/- 66 nM. PACAP was 100-fold more potent than vasoactive intestinal polypeptide (VIP) (PACAP27 shows 68% amino acid sequence homology with VIP), and had a prolonged duration of action, a 1 min exposure to 1 microM PACAP27 lasting 135 +/- 7 min and to 1 microM PACAP38 108 +/- 3 min. Adenylate cyclase activity in homogenates of rabbit aortic smooth muscle cells was increased by PACAP27 and PACAP38 with EC50s of 4.4 and 0.73 nM, respectively. PACAP27 and PACAP38 are potent, long-lasting, endothelium-independent vasodilators.     

Distribution and effects of pituitary adenylate cyclase activating peptide in cat and human lower oesophageal sphincter.

1. The localization, tissue concentrations, and effects of pituitary adenylate cyclase activating peptide (PACAP) 27 and 38 were investigated in cat and human lower oesophageal sphincter (LOS), and compared with those of vasoactive intestinal peptide (VIP) and helospectin. 2. PACAP-immunoreactive nerve structures were found in the cat and human LOS, with an abundance in the circular smooth muscle layer. PACAP 27-immunoreactivity was often co-localized with VIP-immunoreactivity. 3. In cat tissue, PACAP (PACAP 27 plus PACAP 38) concentrations were 50 fold lower than VIP concentrations; in human tissue they were 10 fold lower. 4. PACAP 27, PACAP 38, helospectin I, and VIP induced concentration-dependent relaxations in circular smooth muscle preparations from cat and human LOS. The order of potency was: VIP > helospectin I > or = PACAP 27 > PACAP 38. NG-nitro-L-arginine, scopolamine, or apamin, did not influence the relaxant effects of PACAP 27 or VIP. 5. In cat preparations, both cyclic AMP and cyclic GMP levels were increased after exposure to PACAP 27 and helospectin I, whereas exposure to VIP was followed by an increase in cyclic AMP levels only. In human preparations, there was an increase in cyclic AMP levels without any change in cyclic GMP levels. 6. These results suggest that in the cat and human LOS, PACAP 27 and VIP can occur within the same nerve structures. PACAP 27 has a potent relaxant action, but its functional importance has to be established.     

A CD strategy for the study of polypeptide folding/unfolding

The circular dichroism spectrum of the 20-residue immunogenic peptide from the foot-and-mouth disease virus (VP1; 141-160 of serotype A, subtype 12) was solvent- and temperature-dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left-handed extended helix, the a-helix, and the α-helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D-NMR studies. A new mechanism for the stabilization of left-handed extended helices and destabilization of a-helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X-ray crystallography.     

Two tyrosine residues in the first transmembrane helix of the human vasoactive intestinal peptide receptors play a role in supporting the active conformation

1: We investigated the human vasoactive intestinal polypeptide (VIP) receptors VPAC(1) and VPAC(2) mutated at conserved tyrosine residues in the first transmembrane helix (VPAC(1) receptor Y146A and Y150A and VPAC(2) receptor Y130A and Y134A). 2: [(125)I]-Acetyl-His(1) [D-Phe(2), K(15), R(16), L(27)]-VIP (1-7)/GRF (8-27) (referred to as [(125)I]-VPAC(1) antagonist) labelled VPAC(1) binding sites, that displayed high and low affinities for VIP (IC(50) values and per cent of high affinity binding sites: wild-type, 1 nM (57+/-9%) and 160 nM; Y146A, 30 nM (40+/-8%) and 800 nM; Y150A, 4 nM (27+/-8%) and 300 nM). [R(16)]-VIP behaved as a "super agonist" at both mutated VPAC(1) receptors and the efficacies of VIP analogues modified in positions 1, 3 and 6 were significantly decreased. 3: VIP was less potent at the Y130A and Y134A mutated VPAC(2) receptors (EC(50) 200 and 400 nM, respectively) than at the wild-type VPAC(2) receptor (EC(50) 7 nM). Furthermore, [hexanoyl-His(1)]-VIP behaved as a "super agonist" at the two mutated VPAC(2) receptors, and VIP analogues modified in positions 1, 3 and 6 were less potent and efficient at the mutated than at wild-type VPAC(2) receptors. However, the Y130A and Y134A mutants could not be studied in binding assays. 4: Our results suggest that the conserved tyrosine residues do not interact directly with the VIP His(1), Asp(3) or Phe(6) residues (that are necessary for receptor activation), but stabilize the correct active receptor conformation.     

Conformational consequences of i,i + 3 cystine linkages: nucleation for α-helicity?

Methods to introduce specific secondary structural elements into peptides and proteins are vital for the rational design of peptide and non-peptide drug candidates as well as in the de novo design of proteins. Here the incorporation of a disulfide linkage between cysteine residues spaced three amino acids apart (i, i+3) as a method to induce helicity is examined. Two dodecamer peptides, A-V-S-E-C-Q-L-C-H-D-K-G-NH₂, differing in the chirality of the cysteine at the fifth position (the i position), have been synthesized and conformationally studied both in the linear and cyclized form. This peptide sequence, derived from the N-terminal sequence of parathyroid hormone related protein, does not form helices, even as part of the 1-34 fully active domain of the protein. The four analogs (two cyclic and two linear) were analyzed both in aqueous solution and in the presence of sodium dodecyl sulfate micelles. In aqueous solution the linear peptides display no evidence for secondary structure, while the cyclization induces a turn centered about the cysteine residues. In the presence of micelles the linear form of the peptides adopts bent conformations, containing turns, but results from both NMR and CD provide no evidence of helices. The oxidized l, l -peptide in the micellar solution does not present a well defined conformation, although the presence of one helical turn is evident. The cyclic d, l analog adopts a helical structure (not an α-helix) extending from residue 2 to 9, with non-standard φ, ψ values caused by the presence of the d -amino acid. These results clearly illustrate that the ability of d -Cys(i), Cys(i+ 3) cyclization to initiate helix formation depends greatly on the solvent used. Therefore, any drug-design principle utilizing this modification for helix nucleation must keep the environment in which the peptide is biologically active in mind. © Munksgaard 1997.     

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D 1H NMR and molecular dynamics

Peptides derived from the inhibitor of cAMP-dependent protein kinase, PKI, have been studied by 2D ¹H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala²⁰-Ser²¹] PKI(5-24), and a phosphorylated form of the latter [Ala²⁰-Ser²¹P] PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI (5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly¹⁴-Gly¹⁷ turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp⁹ and Arg¹⁵. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2°C) shows the occurrence of pH-dependent contacts with Phe¹⁰. We conclude that the binding of short inhibitors, such as PKI (5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala²⁰-Ser²¹] PKI (5-24) and the product [Ala²⁰-Ser²¹P] PKI(5-24), give very similar structures with local rearrangements involving some of the side chains. © Munksgaard 1997.     

Pituitary adenylate cyclase-activating polypeptide, helospectin, and vasoactive intestinal polypeptide in human corpus cavernosum.

1. The distribution and effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27 and -38), helospectin (Hel-1 and Hel-2), and vasoactive intestinal polypeptide (VIP), were investigated in isolated preparations of human corpus cavernosum (CC). 2. Immunohistochemistry revealed coinciding profiles of nerve structures that showed immunoreactivities for VIP and PACAP, and VIP and Hel. Confocal microscopy showed the co-existence of VIP- and PACAP-immunoreactivities, and VIP- and Hel-immunoreactivities in most (90%) varicose nerve structures. 3. As determined by radioimmunoassay, the amounts of VIP, PACAP-27, and PACAP-38 in the preparations were 61.7 +/- 11.6, 0.1 +/- 0.05, and 3.7 +/- 0.5 pmol g-1 wet weight of tissue (pmol g-1 wet wt.), respectively. In tissue from patients with diabetes, the content of VIP was lower (13.7 +/- 0.5 pmol g-1 wet wt.), whereas that of PACAP (-27 and -38) was unchanged. 4. Cyclic nucleotide levels were determined in preparations exposed to PACAP-27, PACAP-38, Hel-1, Hel-2, and VIP. All the peptides, but Hel-2, significantly increased the concentrations of cyclic AMP, whereas the levels of cyclic GMP were unchanged. 5. The peptides concentration-dependently relaxed noradrenaline-contracted preparations. The order of potency was VIP > PACAP 27 > Hel-1 > Hel-2 > PACAP-38. 6. Hel-1, VIP and PACAP-27 effectively counteracted electrically induced contractions. At 10(-6) M, the highest peptide concentration used, the inhibitory effects obtained reached 96 +/- 3%, 87 +/- 6%, and 80 +/- 3%, respectively. 7. The results suggest that PACAP and Hel-1 are co-localized with VIP in nerve structures within the human cavernous tissue, and that the peptides are effective relaxants of CC preparations in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of PACAP(6–38) on relaxations induced by PACAP, VIP and non-adrenergic, non-cholinergic nerve stimulation in the guinea-pig taenia caeci

The effect of the pituitary adenylate cyclase activating polypeptide (PACAP) receptor antagonist PACAP(6-38) on the relaxant response to exogenous PACAP, vasoactive intestinal polypeptide (VIP) and nonadrenergic, non-cholinergic (NANC) nerve stimulation was tested in the guinea-pig taenia caeci, in the presence of atropine (10(-6) M) and guanethidine (3x10(-6) M). PACAP(6-38) (3x10(-6) M) strongly inhibited sub-maximal relaxations evoked by exogenous PACAP (1-3x 10(-8) M) or VIP (10(-8) M), but not those due to isoprenaline (4-8x10(-8) M) or ATP (10(-6) M). PACAP(6-38) caused a small but significant (approximately 20%) inhibition of the NANC relaxation due to electrical field stimulation (1 Hz or 10 Hz for 20 s). At these frequencies PACAP(6-38) caused no inhibition of the NANC relaxation in the presence of the P2 purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 5x10(-5) M), or PPADS plus the NO-synthase blocker NG-nitro-L-arginine (L-NOARG; 10(-4) M); in preparations pretreated with L-NOARG (10(-4) M) alone PACAP(6-38) retained its inhibitory effect. The PPADS- and L-NOARG-resistant NANC relaxation with 10 Hz electrical stimulation was blocked by apamin (10(-7) M); it was not significantly modified by the tachykinin receptor antagonist spantide (10(-5) M). Tachyphylaxis to PACAP(1-27) (10(-7) M for 10 min) strongly inhibited the relaxation due to PACAP(1-38) (1-3x10(-8) M) and reduced electrical stimulation-evoked relaxations by half. The putative VIP antagonist VIP(10-28) (10(-5) M) failed to significantly reduce the relaxant action of exogenous VIP (1-3x10(-8) M). Relaxation induced by PACAP(1-38) (1-2x10(-8) M) was not influenced by a mixture of PPADS (5x10(-5) M) and L-NOARG (10(-4) M). It is concluded that: (a) PACAP(6-38) is a VIP/PACAP antagonist in the guinea-pig taenia caeci; (b) a release of a VIP/PACAP-like substance from enteric nerves is involved in the NANC relaxation in this preparation, but its contribution is relatively small and seems to depend on the functional integrity of the PPADS-sensitive inhibitory mechanism; (c) the PPADS- plus L-NOARG-resistant NANC relaxation probably involves apamin-sensitive K+ channels.     

INVOLVEMENT OF PACAP IN ACID-INDUCED HCO3RESPONSE IN RAT DUODENUMS

Pituitary adenylate cyclase activating polypeptides (PACAP) stimulate duodenal HCO₃⁻secretion in the rat. The present study was performed to determine whether endogenous PACAP is involved in the mechanism of acid-induced HCO₃⁻response in the duodenum, using a PACAP antagonist, PACAP6-27. Under urethane anaesthetised conditions, a duodenal loop that was made between the pylorus and the area just above the outlet of the common bile duct was perfused with saline, and the HCO₃⁻secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mmHCl. Duodenal HCO₃⁻secretion was significantly stimulated by i.v. administration of PACAP-27 (8 nmol kg⁻¹) as well as vasoactive intestinal polypeptide (VIP: 8 nmol kg⁻¹). The effect of PACAP-27 (8 nmol kg⁻¹) was equivalent to that induced by prostaglandin E₂(300 μg kg⁻¹, i.v.) and significantly suppressed by either PACAP6-27 (40 nmol kg⁻¹, i.v.) or VIP antagonist (Ac-Tyr¹,d-Phe²-VIP: 40 nmol kg⁻¹, i.v.). These peptide antagonists suppressed duodenal HCO₃⁻secretory response to VIP but did not have any effect on either basal or PGE₂-stimulated HCO₃⁻secretion. On the other hand, the duodenal mucosa responded to acidification by increasing HCO₃⁻secretion in a indomethacin-sensitive manner, and this process was also significantly suppressed by both PACAP6-27 and VIP-antagonist. Duodenal damage induced by acid perfusion (100 mmHCl for 4 h) was significantly worsened by PACAP6-27, VIP antagonist as well as indomethacin at the doses that suppressed acid-induced HCO₃⁻secretion. These findings suggest that PACAP may play a role in local modulation of the duodenal mucosal integrity, by mediating the HCO₃⁻secretory response induced by mucosal acidification.     

氮唑类抗真菌药物药效构象及其与酶活性位点对接

目的： 研究氮唑类抗真菌药物与其受体蛋白活性位点相互作用机理。方法： 用随机构象搜寻和分子动力学模拟退火法确定15个4种不同类型的氮唑类抗真菌药物最低能量构象;用活性类似物法限定药物分子药效基团之间的距离,搜寻到各化合物药效构象;将各化合物药效构象对接到白色念珠菌羊毛甾醇14α去甲基化酶活性位点中。结果： 4种结构类型的氮唑类药物在酶活性位点中有相似的对接位置;真菌和哺乳动物的活性位点结构特异性的残基分布在F螺旋C端、β6-1和β6-2区中;氮唑类抗真菌药物共同的卤代芳环结构落入相同的疏水空穴中,其中Y132的侧链羟苯基结构可与抑制剂卤代芳环形成电子迁移复合物。结论： 对接结果与已知SAR分析结论相符,阐明了氮唑类药物与活性位点的氨基酸残基作用方式,探讨结构选择性药物的结构需求。     

Testing for cis‘ proline with α-aminoisobutyric acid substitution

Substitution of Pro residues with AIB (α-aminoisobutyric acid) residues in peptides provides a means of evaluating the presence of cis' proline conformations both in solution and, using bioassay data, in a receptor complex. ¹ H n.m.r. has been used to probe the DMSO solution conformation of all seven of the possible AIB/Pro isomers of bradykinin. AIB substitution for Pro² and/or Pro³ appears to stabilize a type III β-turn involving the N-terminal residues, but not an incipient 3₁₀ helix suggested by model peptides. These substitutions are correlated with low biological potencies, suggesting that such conformational features may be incompatible with receptor complexation. Alternatively, AIB⁷ -bradykinin analogs exhibit a variety of long range shift perturbations relative to bradykinin. The data suggests that bradykinin can adopt several folded conformations, including β-turns involving both Ser⁶-Pro⁷-Phe⁸-Arg⁹ and Phe⁵-Ser⁶-Pro⁷-Phe⁸. The relatively high biological activities of the AIB⁷-BK suggest that the complexed form of the peptide is characterized by a cis' Pro⁷ conformation.     

Neuronal and smooth muscle receptors involved in the PACAP- and VIP-induced relaxations of the pig urinary bladder neck

BACKGROUND AND PURPOSE: As pituitary adenylate cyclase-activating polypeptide 38 (PACAP 38)- and vasoactive intestinal peptide (VIP) are widely distributed in the urinary tract, the current study investigated the receptors and mechanisms involved in relaxations induced by these peptides in the pig bladder neck. EXPERIMENTAL APPROACH: Urothelium-denuded strips were suspended in organ baths for isometric force recordings and the relaxations to VIP and PACAP analogues were investigated. KEY RESULTS: VIP, PACAP 38, PACAP 27 and [Ala(11,22,28)]-VIP produced similar relaxations. Inhibition of neuronal voltage-gated Ca(2+) channels reduced relaxations to PACAP 38 and increased those induced by VIP. Blockade of capsaicin-sensitive primary afferents (CSPA), nitric oxide (NO)-synthase or guanylate cyclase reduced the PACAP 38 relaxations but failed to modify the VIP responses. Inhibition of VIP/PACAP receptors and of voltage-gated K(+) channels reduced PACAP 38 and VIP relaxations, which were not modified by the K(+) channel blockers iberiotoxin, charybdotoxin, apamin or glibenclamide. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin produced potent relaxations. Blockade of protein kinase A (PKA) reduced PACAP 38- and VIP-induced relaxations. CONCLUSIONS AND IMPLICATIONS: PACAP 38 and VIP relax the pig urinary bladder neck through muscle VPAC(2) receptors linked to the cAMP-PKA pathway and involve activation of voltage-gated K(+) channels. Facilitatory PAC(1) receptors located at CSPA and coupled to NO release, and inhibitory VPAC receptors at motor endings are also involved in the relaxations to PACAP 38 and VIP, respectively. VIP/PACAP receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain*

Abstract: The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well‐known helix‐turn‐helix motif. The recognition helix penetrates the DNA major groove, gives specific protein–DNA contacts and forms direct, or water‐mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42–53, where residues W48 and F49, not involved in the protein–DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well‐defined secondary structure in TFE and in TFE/water (90/10, v/v) with an α‐helix encompassing residues 4–9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted α‐helical conformation involving residues 4–8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the α‐helical conformation even if separated from the rest of the molecule.