Fungal infection of human organs by resistant melanin-synthesizing species is one of the pathogenic factors and one of the real consequences of the accident at Chernobyl power plant

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins. 2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [3H]-inositol phosphates ([3H]-IPs) as a bioassay. 3. Bradykinin (BK) induced a maximal 1.95 +/- 0.24 fold (n = 17) and 2.51 +/- 0.29 fold (n = 26) stimulation of [3H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2-4.5 fold and 2.0-2.9 fold stimulation by histamine (100 microM) and platelet activating factor (100 nM) in both cell-types, respectively. 4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC50 = 3.26 +/- 0.61 nM, n = 18), Lys-BK (EC50 = 0.95 +/- 0.16 nM, n = 5), Met-Lys-BK (EC50 = 2.3 +/- 0.42 nM, n = 5), Ile-Ser-BK (EC50 = 5.19 +/- 1.23 nM, n = 6), Ala3-Lys-BK (EC50 = 12.7 +/- 2.08 nM, n = 3), Tyr8-BK (EC50 = 19.3 +/- 0.77 nM, n = 3), Tyr5-BK (EC50 = 467 +/- 53 nM, n = 4) and des-Arg9-BK (EC50 = 14.1 +/- 2.7 microM, n = 4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC50 = 2.02 +/- 0.69 nM (n = 7), Tyr8-BK, EC50 = 14.6 +/- 2.7 nM (n = 3), Tyr5 = BK, EC50 = 310 +/- 70 nM (n = 4) and des-Arg9-BK, EC50 = 12.3 +/- 3.8 microM (n = 3). 5. The bradykinin-induced responses were competitively antagonized by the B2-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK; Icatibant; molar antagonist potency = 2.9 nM; pA2 = 8.54 +/- 0.06, n = 4; and slope = 1.04 +/- 0.08) and D-Arg0[Hyp3,Thi5,8, DPhe7]-BK (KB = 371 nM; pKB = 6.43 +/- 0.08, n = 4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4 +/- 1.8 nM (pKi = 8.11 +/- 0.12, n = 4), this being similar to the potency observed in the immortalized cells. 6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [3H]-IPs by the specific B2-receptor antagonists, strongly suggests that a B2-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells. 7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B2-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.     

Effects of the prolonged administration of bradykinin on the rat pituitary-adrenocortical axis

The effects of a prolonged administration of bradykinin (BK) and/or D-Arg, [Hyp3, D-Phe7]-BK, a specific antagonist of BK receptors (BK-A) (daily subcutaneous injections of 4 nmol/rat for 6 days) on the function of the pituitary-adrenocortical axis were investigated. BK did not change plasma aldosterone concentration (PAC), but markedly lowered that of corticosterone (PBC) and consequently induced a compensatory hypersecretion of ACTH by the pituitary gland. BK-A did not apparently affect the function and growth of the adrenal gland, but, when administered together with BK, markedly raised both PAC and PBC, and provoked a significant atrophy of the adrenal gland, probably due to loss of parenchymal cells. Taken together, these rather puzzling findings do not appear to provide clear evidence for the involvement of BK in the physiological regulation of adrenocortical growth and steroidogenic capacity in rats.     

Various glucocorticoids differ in their ability to induce gene expression, apoptosis and to repress NF-kappaB-dependent transcription

1. The nonpeptide bradykinin (BK) B2 receptor antagonist, FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methylquinoline), and agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridyl methoxy)quinoline) have been identified. These compounds have a common chemical structure, and the 2-pyridylmethoxy group is the only structural difference between them. 2. Both FR165649 and FR190997 displaced [3H]-BK binding to B2 receptors in guinea-pig ileum membranes, with an IC50 of 4.7 x 10(-10) M and 1.5 x 10(-9) M, respectively. They also displaced [3H]-BK binding to B2 receptors in human lung fibroblast IMR-90 cells, with an IC50 of 1.6 x 10(-9) M and 9.8 x 10(-10) M, respectively. 3. In guinea-pig isolated ileum-preparations, FR165649 had no agonistic effect on contraction and caused parallel rightward shifts of the concentration-response curves to BK on contraction. Analysis of the data produced a nominal pA2 value of 9.2+/-0.1 (n=5) and a slope of 1.4+/-0.1 (n=5). On the other hand, FR190997 induced concentration-dependent contraction of guinea-pig ilea with a pD2 of 7.9+/-0.2 and the contraction was inhibited by a specific peptide bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK) in a non-competitive manner. 4. In IMR-90 cells, FR165649 had no agonistic effect on phosphatidyl inositol (PI) hydrolysis and caused parallel rightward shifts (approximately 200 fold shift at 10(-7) M) of the concentration-response curves to BK on PI hydrolysis. FR190997 induced concentration-dependent PI hydrolysis in IMR-90 cells with a pD2 of 8.4+/-0.1, and this effect was inhibited by Hoe 140. 5. These results indicate that FR165649 and FR190997 are, respectively, a potent bradykinin B2 receptor antagonist and agonist, and that the agonistic activity depends on the small part of the nonpeptide ligand. FR165649 and FR190997 may be useful tools for studying the relationship between ligands and receptors.     

The tachykinin NK1 receptor antagonist, RP67580, inhibits the bradykinin-induced rise in intracellular Ca2+ concentration in bovine pulmonary artery endothelial cells

The bradykinin-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and the bradykinin receptor involved in this response were characterized in bovine pulmonary artery endothelial cells. It was found that bradykinin induces an intracellular biphasic Ca2+ response, consisting of a transient peak followed by an elevated plateau phase. Both bradykinin and the bradykinin B1 receptor agonist, des-Arg9-bradykinin, induced a concentration-dependent increase in [Ca2+]i, but the bradykinin-induced rise was much greater. Moreover, the bradykinin-induced [Ca2+]i rise could be inhibited by the bradykinin B2 receptor antagonists, D-Arg0[Hyp3, Thi(5,8), D-Phe7]bradykinin and Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]bradykinin), but not by the bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin. From these results it can be concluded that a bradykinin B2 receptor is involved in this response. Furthermore, we found that the tachykinin NK1 receptor antagonist, RP67580 ([imino 1 (methoxy-2-phenyl)-2 ethyl]-2 diphenyl 7,7 perhydroisoindolone-4 (3aR, 7aR)), and its negative enantiomer, RP68651 (2-[1-imino 2-(2 methoxy phenyl) ethyl] 7,7 diphenyl 4-perhydroisoindolone (3aS-7aS)), could inhibit the bradykinin-induced [Ca2+]i response, although no functional tachykinin NK1 receptors were found. Binding studies evidenced no binding of RP67580 or RP68651 to the bradykinin receptor. We conclude that RP67580 inhibits the bradykinin-induced rise in [Ca2+]i via a bradykinin B2 receptor-independent mechanism.     

NMR and CD conformational studies of bradykinin and its agonists and antagonists: application to receptor binding

Most physiological processes are regulated by peptides that perform their functions by interacting with specific receptors on cells. Specific conformations of the peptides are required for correct interactions to take place, and a knowledge of the biologically important conformation is vital for the understanding of biological function. Over the last few years extensive studies using nuclear magnetic resonance and circular dichroism have been carried out on bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and its antagonists with the objective of developing new drugs to combat severe pathologies associated with its production. In the present review, these techniques for the determination of peptide conformation are reviewed and applied to the study of bradykinin and its antagonists. Modeling of these conformational data in the presence of the B2 receptor or an antibody allows the biologically active conformations to be deduced and these are presented in this review.     

Degradation of bradykinin in semen of ram and boar

The pattern of bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9)-inact iva ting peptidases in semen of boar and ram was investigated. The degradation of BK in semen was completely abolished by the metalloprotease inhibitors EDTA and o-phenanthroline. Inhibitors of angiotensin-converting enzyme (ACE; EC 3.4.15.1) and phosphoramidon, an inhibitor of neutral metalloendopeptidase (NEP; EC 3.4.24.11), were only partially effective in preventing BK degradation in semen. An additive effect was seen with simultaneous inhibition of both enzymes, resulting in complete abolition of BK degradation. HPLC analysis demonstrated that exogenous BK in semen is cleaved at Gly4-Phe5, Phe5-Ser6 and Pro7-Phe8. These results indicate that NEP and ACE are the main peptidases responsible for rapid BK inactivation in semen. The involvement of other peptidases known to be responsible for BK cleavage in other tissues and body fluids, namely carboxypeptidase N (EC 3.4.12.7), post proline cleaving enzyme (EC 3.4.21.26) and aminopeptidase P (EC 3.4.11.9) was excluded. NEP and ACE were shown to be localized mainly in seminal plasma and to a lesser extent on sperm cells.     

Protein kinase CK2: biphasic kinetics with peptide substrates

1. The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK. 2. The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) > or = BK (pD2 8.60) > [Aib7]BK (pD2 6.38) > desArg9BK and LysdesArg9BK (inactive). 3. Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive. 4. Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4-6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists. 5. When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60) > desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive. 6. The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK. 7. The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea-pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N-terminal Lys.     

Involvement of endogenous kinins in the pathogenesis of peptidoglycan-induced arthritis in the Lewis rat

OBJECTIVE: To investigate the pathophysiologic roles of endogenous bradykinin (BK) and des-Arg9-BK on local and systemic inflammatory responses in a rat model of acute arthritis induced by peptidoglycan-polysaccharide (PG-APS). METHODS: Female Lewis rats were injected intraperitoneally with PG-APS. Selective antagonists of B1 (Lys-[Leu8]-des-Arg9-BK) and B2 (Hoe 140) receptors were infused at 500 microg/kg and 5 mg/kg per day for 6 days, starting 3 days before induction of inflammation, with subcutaneous micro-osmotic pumps. The local inflammatory response was assessed by paw edema, joint swelling, and tissue content of BK and des-Arg9-BK. These peptides were measured by highly sensitive and specific chemiluminescent enzyme immunoassays. Systemic inflammatory reaction was evaluated by the hepatic concentration of the type 2 acute-phase protein T-kininogen. RESULTS: PG-APS induced significant paw edema and joint swelling 24-72 hours after intraperitoneal injection. The maximal responses to PG-APS observed at 72 hours were significantly reduced (31-38%) by the combination of both B1 and B2 receptor antagonists at 5 mg/kg per day. PG-APS induced a significant increase of BK (up to 5.3-fold) and des-Arg9-BK (up to 4.1-fold) 72 hours after challenge. Liver T-kininogen content was increased by 5.3-, 7.7-, and 5.8-fold at 24, 48, and 72 hours, respectively, after PG-APS injection. At 24 hours, Hoe 140 and Lys-[Leu8]-des-Arg9-BK increased liver T-kininogen content by 43% and 45%, respectively, but they had no effect at 72 hours. CONCLUSION: The results indicate that endogenous kinins are involved in local and systemic acute inflammatory responses, through both B1 and B2 kinin receptors, in the model of PG-APS-induced arthritis.     

Rabbit brain thiol-activated endopeptidase. Hydrolysis of bradykinin and kininogen

A neutral brain endopeptidase which hydrolyzes bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) at the Phe5-Ser6 peptide bond was activated about 10 times by dithiothreitol. The preferential specificity of the enzyme for small peptides was suggested on the basis of the absence of activity toward a bradykinin-related protein such as S-carboxymethylated plasma kininogen.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.     

Nitric oxide as mediator of bradykinin-induced pancreatic circulatory and metabolic responses

Bradykinin (BK) is an endogenous nonapeptide with potent vasodilator properties of the visceral circulation. BK alters vascular tone via two BK receptor subtypes, B1 and B2. Current experimental evidence suggests that the dilator action of BK in some vessels is mediated primarily by B2 receptor activation. In addition, there are reports that BK increases endothelial generation of vasodilator factors, such as nitric oxide (NO). The present study had two aims. First, to explore the role of BK-receptors in the pancreatic vasodilatatory and metabolic responses to BK. Second aim was to examine whether endogenous NO play a role in the mediation of BK-receptors induced pancreatic circulatory and metabolic activity. In anesthetized dogs, the superior pancreatico-duodenal artery blood flow (SPBF) was measured by ultrasonic blood flowmeter (Transonic System T-206), pancreatic microcirculatory blood flow (PBF) was determined by laser Doppler flowmetry (Periflux 4001 Master). Pancreatic oxygen consumption (PVO2) was calculated as the product of the arteriovenous oxygen difference (AVO2) across the pancreatic circulation and SPBF. Drugs were infused into the superior pancreatico-duodenal artery. BK (0.01-1.0 mg/kg/min) increased maximally SPBF by 180 +/- 15%, PBF by 208 +/- 22% and PVO2 by 145 +/- 11%, respectively. Pretreatment with B2-subtype receptor antagonist, D-Arg, [Hyp3, Thi5,8, D-Phe7] BK inhibited significantly BK-induced increase in SPBF, PBF and PVO2 by 86 +/- 8%, 73 +/- 7% and 85 +/- 6%, respectively. A nitric oxide synthesis inhibitor (L-NNA) administered i.v. at dose of 25 mg/kg 20 min before BK, inhibited significantly the pancreatic hyperemic and metabolic responses. The results presented emphasize an important role of B2 receptors in the mediation of pancreatic circulatory and metabolic responses to bradykinin. Endogenous NO plays a mediatory role in the pancreatic vascular and metabolic responses due to stimulation of B2-receptors.     

B1 receptor involvement in the effect of bradykinin on venular endothelial cell proliferation and potentiation of FGF-2 effects

1. Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. 2. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. 3. [3H]-thymidine incorporation in subconfluent and synchronised CVEC was used to monitor DNA synthesis over 24 h. BK promoted a concentration-dependent increase of DNA synthesis with maximal activity at 100 nM. At this concentration BK also induced 18 fold accumulation of c-Fos protein immunoreactivity in the nucleus within 1 h from peptide exposure. 4. The total number of cells recovered after 48 h exposure to BK was increased in a concentration-dependent manner. Maximal effect was produced by 100 nM concentration of the peptide which produced 50% increase in cell number. The selective B1 receptor agonist Des-Arg9-BK mimicked the proliferative effect of BK, while the B2 receptor agonist kallidin was devoid of any activity. The proliferation induced by BK was abolished in a concentration-dependent manner by the addition of the B1 selective antagonist Des-Arg9-Leu8-BK, while the selective B2 receptor antagonist HOE140 did not modify BK-induced growth. 5. DNA synthesis and growth promoted by a threshold concentration of fibroblast growth factor-2 (FGF-2) (0.25 nM) were potentiated by increasing concentrations of BK and Des-Arg9-BK. 6. Endothelial cell migration assessed by the Boyden Chamber procedure was not promoted by BK or the selective B1 and B2 receptor agonists. 7. These data are the first demonstration that BK promotes growth of endothelial cells from postcapillary venules. The mitogenic activity of BK involves c-Fos expression and potentiates the growth promoting effect of FGF-2. Only the B1 receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules.     

Novel cyclic analogs of angiotensin II with cyclization between positions 5 and 7: conformational and biological implications

To study the conformational features of molecular recognition of angiotensin II (Asp1-Arg2-Val3-Tyr4-Val/IIe5-His6-Pro7-Phe8, AII), the synthesis and biological testing of several cyclic analogs of AII cyclized between positions 5 and 7 have been performed. The synthesized analogs were Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Pen7)-Phe8 (3), Sar1-Arg2-Val3-Tyr4-cyclo(Asp5-His6-Apt7)-Phe8 (4), Sar1-Arg2-Val3-Tyr4-cyclo(Glu5-His6-Apt7)-Phe8 (5), Sar1-Arg2-Val3-Tyr4-cyclo-(Cys5-His6-Mpt7)-Phe8 (6), Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Mpc7)-Phe8 (7), Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpt7)-Phe8 (8), and Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpc7)-Phe8 (9), where Apt stands for 4-amino-trans-proline, and Mpt and Mpc for 4-mercapto-trans- and -cis-prolines, respectively. Compound (9) showed good affinity at AT-1 receptors, namely a KD = 20 nM. In functional assays, it showed the characteristics of a weak partial agonist with a relative affinity of 0.26% of that for AII and an intrinsic efficacy, alpha E, of 0.42. Molecular modeling suggested a possible explanation for this finding: the relatively strong binding and the weak partial agonistic activity of compound 9 are due to interaction with AT-1 receptor of only two functionally important groups, namely, the side chains of the His6 and Phe8 residues.     

Structural model of a cyclic dynorphin A analog bound to dodecylphosphocholine micelles by NMR and restrained molecular dynamics

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.     

Development of a new method for simultaneously evaluating mucociliary clearance and pulmonary epithelial permeability in rabbit experiments by means of 18FDG, three-dimensional positron emission tomography and rectilinear scan

OBJECTIVE: To determine which types of kinin receptor are present in human bronchial epithelial cells we studied the capability of bradykinin to mobilize intracellular Ca2+ ([Ca2+]i) in a human bronchial epithelial cell line (16HBE cells). MATERIAL: Human bronchial epithelial cell line transformed with an original defective simian virus 40 (SV40). TREATMENT: Bradykinin (0.1 pM to 0.1 microM), des-Arg9 bradykinin (1 microM), des-Arg10) kallidin (1 microM), indomethacin (1 microM), phosphoramidon (1 microM), captopril (1 microM), des-Arg9-[Leu8]bradykinin (1 microM), HOE 140 (DArg-[Hyp3, Thi5, DTic , Oic8]-bradykinin) (1 microM), and NPC 16731 (DArg-[Hyp3, Thi5, DTic7, Tic8]-bradykinin) (1 microM). METHODS: The mobilization of [Ca2+]i was determined by the fura-2 method. Two sample Wilcoxon rank-sum (Mann-Whitney) test was used for statistical calculations. RESULTS: Bradykinin, but not the selective agonists for kinin B1 receptor des-Arg9 bradykinin and des-Arg10 kallidin, increased the mobilization of [Ca2+]i (EC50, 0.079+/-0.009nM) in 16HBE cells in a concentration-dependent manner. Pretreatment with the cyclooxygenase inhibitor indomethacin (1 microM) or the peptidase inhibitors, phosphoramidon (1 microM) or captopril (1 microM), did not affect the response to bradykinin. The kinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin (1 microM), was inactive. HOE 140 and NPC 16731, two selective antagonists of the kinin B2 receptor abolished the response to bradykinin (IC50 of HOE 140 and NPC 16731 were 0.52+/-0.037nM and 1.67 +/- 0.41 nM, respectively). CONCLUSIONS: The present data indicate the presence of kinin B2 receptors in the 16HBE cells.     

I1-imidazoline receptors and cholinergic outflow to the airways

The modulatory effect of bradykinin on electrically-induced noradrenaline release was assessed in isolated atria from normal and B2 knockout transgenic mice preincubated with [3H]noradrenaline. Concentrations of 1, 3 and 10 nM of bradykinin did not significantly alter the outflow of radioactivity whereas higher concentrations of bradykinin (30 and 100 nM) enhanced it. The facilitatory effect of 30 nM bradykinin was inhibited by a selective bradykinin B2 receptor antagonist. Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, 30 nM), and by a protein kinase C inhibitor, bisindolylmaleimide (1 microM). The co-administration of bradykinin (1 to 100 nM) with either [Leu8]des-Arg9-bradykinin (100 nM), AcLys[DbetaNal7,Ile8]des-Arg9-bradykinin (30 nM) (bradykinin B1 receptor antagonists) or diclofenac (1 microM) (a cyclooxygenase inhibitor), shifted the facilitatory effect of bradykinin to lower concentrations. The facilitatory effect of bradykinin also was enhanced by enalaprilat (1 microM) and mergetpa (1 microM), inhibitors of angiotensin-converting enzyme (kininase II) and kininase I, respectively. In contrast, selective bradykinin B1 receptor agonists, des-Arg9-bradykinin (1 to 100 nM) and Sar[D-Phe8]des-Arg7-bradykinin (1 to 100 nM), did not significantly affect the stimulation-induced outflow of radioactivity. Neither bradykinin (100 nM) nor des-Arg9-bradykinin (100 nM) had any modulatory effect in B2 knockout transgenic mice. These findings suggest that the facilitatory effect of bradykinin on noradrenaline release in the mouse atria is mediated exclusively by presynaptic bradykinin B2 receptors which are linked to protein kinase C. The greater release of noradrenaline with bradykinin under inhibition of prostaglandins production and kininases I and II activity might be of importance in pharmacotherapies.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.     

Treatment of severe peri-implant bone loss using autogenous bone and a resorbable membrane. Case report and literature review

The objective of this study was to elucidate the solution conformation of cyclo-(1,12) Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Ar g9-Gly10-Gly11-Cys12 (1) derived from the intercellular adhesion molecule-1 (ICAM-1). Cyclic peptide 1 inhibits homotypic adhesion of T-cells (Molt-3) mediated by ICAM-1 and the leukocyte function-associated antigen-1 (LFA-1) on the surface of T-cells. Cyclic peptide 1 is more potent than is the linear peptide Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Ar g9-Gly10-Gly11-Cys12 (2) in inhibiting homotypic adhesion. The difference in biological activity of peptides 1 and 2 may be due to the more stable conformation of cyclic peptide 1 compared to linear peptide 2 or because cyclization prevents the peptide from adopting non-productive conformation. Therefore, conformational studies of cyclic peptide 1 will give a better understanding of its biological active conformation. The conformational studies of cyclic peptide 1 were done by NMR, CD and molecular dynamics simulations. NMR studies indicated that the major conformation of cyclic peptide 1 contained trans-configuration at both X-Pro peptide bonds. Type I beta-turns at Lys4-Val5-Ile6-Leu7 and Leu7-Pro8-Arg9-Gly10 were found in cyclic peptide 1. The C- and N-terminal regions of this peptide were stabilized by antiparallel beta-sheet-like structure with the presence of intramolecular hydrogen bonds. The overall structure of this peptide exposed the hydrophobic side chains on one face of the molecule and the hydrophilic side chains on the other.     

Conformational analysis of two cyclic analogs of angiotensin: implications for the biologically active conformation

Conformations of two cyclic analogs of angiotensin (Asp1-Arg2-Val3-Tyr4-Val/Ile5-His6-Pro7-Phe8, AT), cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, were studied, independently employing two complementary techniques, energy calculations and NMR measurements in DMSO solution. NMR data were indicative of well-defined solution conformations for the cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, including the phi values for the Cys3/HCys3 and Tyr4 residues, as well as the chi 1 value for the Tyr4 residue. Solution conformations for the exocyclic linear parts of both molecules cannot be described by the NMR data with the same precision. At the same time, independent energy calculations revealed the same conformations of cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT among low-energy conformers for both peptides. Moreover, the same conformations are compatible with the model of AT receptor-bound conformation (Nikiforovich & Marshall, 1993), which assumes the particular spatial arrangement of aromatic moieties of Tyr4, His6, and Phe8 residues and the C-terminal carboxyl. These conformers of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT contain "an open turn" in the backbone of the Tyr4-Val5 residues, instead of the earlier proposed beta-like reversal, thus confirming the suggestion that the conformation(s) ensuring binding of AT analogs with specific receptors should not be described in terms of a unique backbone conformer.     

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.