Production of arachidonic acid metabolites by endothelial cells in hyperoxia

This study investigated the response of bovine pulmonary artery endothelial cells to incubation in hyperoxia (95% O2-5% CO2). Changes in cell number and morphology, release of lactate dehydrogenase, and production of arachidonic acid metabolites were assessed during continuous exposure of confluent endothelial monolayers to air (air-5% CO2, "controls") or O2 (95% O2-5% CO2, "O2-exposed") for periods of 12-72 h. Control monolayer cell numbers remained constant (approximately 2,000,000 cells/flask), whereas the number of cells in O2-exposed monolayers decreased progressively to 30% of controls (P less than 0.01) by 72 h. As assessed by radioimmunoassay, both control and O2-exposed cells produced the prostacyclin metabolite, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin F2 alpha (PGF2 alpha), but no thromboxane metabolite (TxB2) was detected. The O2-exposed cells released significantly more 6-keto-PGF1 alpha and PGF2 alpha than control cells when apparent net production rates over the entire 72-h period were compared. In addition, both control and O2-exposed (48 h) endothelial monolayers released immunoreactive leukotriene B4 (LTB4) on stimulation with calcium ionophore (10 microM A23187). As with the cyclooxygenase products, O2-exposed cells released more immunoreactive LTB4 than did controls. Both cyclooxygenase and lipoxygenase metabolites of arachidonic acid are released by cultured endothelial cells during the development of O2 toxicity.     

Receptor-Linked Hydrolysis of Phosphoinositides and Production of Prostacyclin in Cerebral Endothelial Cells

The receptor agonist-mediated hydrolysis of phosphoinositides and production of prostacyclin were studied in murine cerebral endothelial cells (MCEC). Of 11 neurotransmitters and neuromodulators examined, carbachol, noradrenaline (NE), bradykinin, and thrombin significantly increased 3H-inositol phosphate accumulation in the presence of LiCl (20 mM). The maximal stimulation of [3H]inositol monophosphate ([3H]IP1) reached approximately 11, 11, seven, and four times the basal levels for carbachol, NE, bradykinin, and thrombin, respectively. The EC50 values of IP1 accumulation for carbachol and NE were 34 and 0.16 microM, respectively. The muscarinic antagonists, atropine and pirenzepine, blocked the carbachol-induced IP1 accumulation with Ki values of 0.3 and 30 nM, respectively. The adrenergic antagonist, prazosin, blocked NE-induced IP1 accumulation with a Ki of 0.1 nM. The calcium ionophore A23187, histamine, glutamate, vasopressin, serotonin, platelet activating factor, and substance P did not stimulate IP1 accumulation. A23187, bradykinin, and thrombin stimulated prostacyclin release to approximately four, four, and two times the basal levels, respectively, whereas carbachol and NE had little effect upon prostacyclin release. These results suggest that the activation of phospholipase C and of phospholipase A2 in MCEC are regulated separately.     

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.     

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: Release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D₄ (LTD₄) or leukotriene C₄ (LTC₄) resulted in concentration dependent increases in prostacyclin release measured as 6-ketoprostaglandin F_1α. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B₄ and leukotriene E₄ (LTE₄) were inactive at con- centrations up to 10 μM. The induction of prostacyclin synthesis by LTC₄ and LTD₄ was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23107 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

Correlation between thrombin-induced prostacyclin production and inositol trisphosphate and cytosolic free calcium levels in cultured human endothelial cells

Cultured human umbilical vein endothelial cells (HUVEC) stimulated with thrombin are known to synthesize prostacyclin at least in part from arachidonate released by phospholipase A2, an enzyme directly activated by calcium. In this study, thrombin stimulation of Quin 2-loaded HUVEC caused rapid and dose-dependent rises in inositol trisphosphate (IP3) and cytosolic free calcium (Ca2+i) levels which preceded a similarly dose-dependent rise in prostacyclin production measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay (ED50 = 0.6-0.7 units/ml for all three effects). Thrombin induced these effects in the absence of extracellular calcium (EGTA) or in the presence of either 8-bromo-cAMP or the calmodulin inhibitor W7. Thrombin inactivated with either diisopropyl fluorophosphate or D-Phe-Pro-Arg-chloromethyl ketone was inactive. In contrast, Quin 2-loaded cultured bovine aortic endothelial cells failed to respond to thrombin, although stimulation with trypsin elevated IP3 and Ca2+i levels and increased 6-keto-PGF1 alpha production. Restimulation of HUVEC with thrombin or histamine 5 min after an initial stimulation with thrombin (2 units/ml for 5 min) failed to induce a second rise in either IP3 or Ca2+i levels or further production of 6-keto-PGF1 alpha, whereas restimulation with ionomycin in the presence or absence of extracellular calcium elevated Ca2+i levels and induced further 6-keto-PGF1 alpha production. However, if the initial stimulation with thrombin was terminated by addition of D-Phe-Pro-Arg-chloromethyl ketone within 10-60 s, restimulation with a second dose of thrombin induced second rises in both IP3 and Ca2+i levels and additional 6-keto-PGF1 alpha production that were greatest when the initial thrombin stimulus was briefest. These results are consistent with the conclusion that IP3 acts as a second messenger by which thrombin elevates Ca2+i levels and initiates prostacyclin synthesis in HUVEC and that in vivo endothelial cells may be stimulated multiple times to synthesize prostacyclin if each period of stimulation is brief.     

Nonpeptide antagonists for kinin receptors

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

NMR and simulated annealing investigations of bradykinin in presence of polyphenols

Epidemiological studies have shown that the incidence of some cardiovascular degenerative diseases appears to be lower in populations with regular but moderate drinking of red wine rich in polyphenols. One of the most important properties of polyphenols is to form complexes with proteins. The linear nonapeptide hormone bradykinin (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH) is involved in a variety of physiological processes such as the cardiovascular processes. Thus, the goal of this work was to study the effects of tannins on the peptide structure by NMR investigations and molecular modeling. The results of these investigations show that in the presence of catechin, the peptide conformation is not affected and is in a random coil structure. On the contrary, the peptide structure is modified by the addition of dimeric proanthocyanidin B3 (catechin 4alpha-->8 catechin). The dimer leads to the formation of a large flexible turn between the 6-9 residues. Thus, the biological activities of bradykinin in the presence of polyphenols could be affected.     

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin.     

Beta-turns induced in bradykinin by (S)-alpha-methylproline.

The ability of (S)-alpha-methylproline (alpha-MePro) to stabilise reverse-turn conformations in the peptide hormone bradykinin (BK = Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) has been investigated. Two BK analogues containing alpha-MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D-ROESY spectra indicative of reverse-turn conformations at both Pro2-Phe5 and Ser6-Arg9, whose formation appears to be cooperative. Some potential applications of alpha-MePro as a reverse-turn mimetic in the construction of synthetic peptide libraries is discussed.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D4 (LTD4) or leukotriene C4 (LTC4) resulted in concentration dependent increases in prostacyclin release measured as 6-keto-prostaglandin F1 alpha. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment, with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B4 and leukotriene E4 (LTE4) were inactive at concentrations up to 10 microM. The induction of prostacyclin synthesis by LTC4 and LTD4 was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23187 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

6-Ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and thromboxane B2 production by endothelial cells, smooth muscle cells and fibroblasts cultured from piglet aorta

After [3H]arachidonic acid labeling, cyclooxygenase products were qualitatively analysed in the media of each cultured vascular cell type by reverse-phase high-performance liquid chromatography (rp-HPLC). The prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha and thromboxane B2 detected in the rp-HPLC radioactive profile were then quantified by radioimmunoassay (RIA) in separate sets of experiments. In preconfluent endothelial cells prostaglandin F2 alpha and 6-ketoprostaglandin F1 alpha were detected in equal amounts (49%), whereas after confluence 6-ketoprostaglandin F1 alpha represented 57% of total secretion (P less than 0.05). Smooth muscle cells secreted mainly prostaglandin F2 alpha (48%) and fibroblasts prostaglandin E2 (44%). Using the bioassay method, antiaggregatory activity was detected only in endothelial cells, though a small percentage of immunoreactive 6-ketoprostaglandin F1 alpha was encountered in smooth muscle cells and fibroblasts (13 and 10%, respectively). Radioimmunological analysis after rp-HPLC separation of the medium of endothelial cells showed that the anti-6-ketoprostaglandin F1 alpha antibody recognized, among other substances, an unidentified compound. Its retention time was similar to that of prostaglandin F2 alpha. This unidentified compound was not detected in the media from smooth muscle cells and fibroblasts.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase

Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP(3))-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP(3) receptor (1-10 microM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 microM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 microM ML-7) or of calmodulin kinase kinase (26 microM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 microM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 microM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.     

Endothelial thromboxane receptors: biochemical characterization and functional implications

We have identified thromboxane specific receptors in membrane preparations of bovine pulmonary artery endothelial cells using a potent thromboxane specific antagonist, [125I]-PTA-OH in a binding assay. The binding was specific and saturable. Neither thromboxane B2, prostaglandin D2 nor prostaglandin F2 alpha displaced the ligand (0.1 nM) at concentrations up to 10 microM. However, binding was displaced by IPTA-OH greater than SQ29548 greater than U46619. In addition, we observed that thromboxane mimetic U46619 significantly lowered the basal production of prostacyclin and also markedly suppressed bradykinin-stimulated prostacyclin released by endothelial cells. We propose that an important biological effect of thromboxane on vascular endothelial cells may be the suppression of prostacyclin production.     

Neuropeptide-Metabolizing Peptidases in Neuro-2a Neuroblastoma and C6 Glioma Cells

Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation.     

Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: fluorescence study

The peptide hormone bradykinin (BK) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its shorter homolog BK(1-5) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)) were labeled with the extrinsic fluorescent probe ortho-aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH(2) and Abz-BK(1-5)-NH(2)). The fragment des-Arg(9)-BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg(9)-BK-DAP(Abz)-NH(2). The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide-lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy-Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK(1-5)-NH(2) presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer.     

In SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors

In mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), acetylcholine and the calcium ionophore A-23187 release endothelium-derived contracting factors (EDCFs), cyclooxygenase derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine are most likely prostacyclin and prostaglandin (PG)H(2), whereas those released by A-23187 remain to be identified. Isometric tension and the release of PGs were measured in rings of isolated aortas of WKY and SHR. A-23187 evoked the endothelium-dependent release of prostacyclin, thromboxane A(2), PGF(2alpha), PGE(2), and possibly PGH(2) (PGI(2) > thromboxane A(2) = PGF(2alpha) = PGE(2)). In SHR aortas, the release of prostacyclin and thromboxane A(2) was significantly larger in response to A-23187 than to acetylcholine. In response to the calcium ionophore, the release of thromboxane A(2) was significantly larger in aortas of SHR than in those of WKY. In both strains of rat, the inhibition of cyclooxygenase-1 prevented the release of PGs and the occurrence of endothelium-dependent contractions. Dazoxiben, the thromboxane synthase inhibitor, abolished the A-23187-dependent production of thromboxane A(2) and inhibited by approximately one-half the endothelium-dependent contractions. U-51605, an inhibitor of PGI synthase, reduced the release of prostacyclin elicited by A-23187 but induced a parallel increase in the production of PGE(2) and PGF(2alpha), suggestive of a PGH(2) spillover, which was associated with the enhancement of the endothelium-dependent contractions. These results indicate that in the aorta of SHR and WKY, the endothelium-dependent contractions elicited by A-23187 involve the release of thromboxane A(2) and prostacyclin with a most likely concomitant contribution of PGH(2).     

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).