Low Concentrations Of Bradykinin Research Articles

ОСОБЛИВОСТІ БРАДИКІНІН-ІНДУКОВАНОГО СКОРОЧЕННЯ ГЛАДЕНЬКИХ М'ЯЗІВ ШЛУНКУ ЗАЛЕЖНО ВІД КОНЦЕНТРАЦІЇ СПОЛУК НА ОСНОВІ 3-ЗАМІЩЕНИХ 1,4-БЕНЗДІАЗЕПІН-2-ОНІВ

The effect of compounds based on 3-substituted-1,4-benzodiazepine-2-ones on contractile activity of smooth muscles of the rat's stomach was analyzed. Action substances MX-1626, MX-1775 for the smooth muscle contraction of like competitive inhibitor of bradykinin – des-Arg9- [Leu8]-Bradykinin acetate, which is observed as increase normalized rate of contraction with increasing of bradykinin concentration and characterized by a slowdown in the first phase of contraction. The most effective 3-subtituted 1,4-benzodiazepin-2-ones was at low concentrations of bradykinin, increasing it concentration their effect is reduced.

Endothelium-Dependent Relaxation in Response to Low Concentrations of Bradykinin Is Enhanced by Phosphoramidon, Bosentan and BQ-123 in Bovine Coronary Arteries In Vitro

An endothelin (ET)-converting enzyme inhibitor phosphoramidon (10 microM), an ET(AB)-receptor antagonist bosentan (10 microM) and an ET(A)-receptor antagonist BQ-123 (1 microM) potentiated endothelium-dependent relaxation of bovine coronary arteries in response to bradykinin (BK) at femtomolar to picomolar concentrations, but not at nanomolar concentrations. BQ-788 (3 microM), an ET(B)-receptor antagonist, showed no significant effects on fM-nM BK-induced relaxation. These results suggest that the endothelium-dependent relaxation of isolated bovine coronary arteries induced by very low concentrations of BK is partly regulated by a complex mechanism involving the ET(A)-receptor antagonism.

Concentration-dependent effects of bradykinin on leukocyte recruitment and venular hemodynamics in rat mesentery.

The results of several recent studies indicate that bradykinin protects tissues against the deleterious effects of ischemia-reperfusion (I/R). However, other studies indicate that bradykinin can act as a proinflammatory agent, inducing P-selectin expression, the formation of chemotactic stimuli, and endothelial barrier disruption. In the present study, we used intravital microscopic techniques to examine the dose-dependent effects of bradykinin on leukocyte-endothelial cell interactions, the formation of platelet-leukocyte aggregates, and venular hemodynamics in rat mesentery in an attempt to explain these divergent findings. Superfusion of the mesentery with low concentrations of bradykinin (</=10(-7) M) increased venular erythrocyte velocity (V(RBC)) without increasing the number of adherent leukocytes, whereas higher concentrations (>/=10(-6) M) decreased V(RBC), increased the number of platelet-leukocyte aggregates, and induced leukocyte adhesion in single postcapillary venules. The formation of platelet-leukocyte aggregates and increased leukocyte adhesion induced by high-dose bradykinin were attenuated by administration of a B(2)-receptor (HOE-140) or a platelet-activating factor (PAF, WEB-2086) antagonist. Thus these adhesive interactions induced by high-dose bradykinin appear to be mediated by a mechanism that is dependent on B(2)-receptor activation and the formation of PAF or PAF-like lipids. The effects of bradykinin on venular V(RBC) and blood flow were also concentration dependent, with low doses producing nitric oxide-mediated vasodilation, whereas high doses decreased V(RBC) by a mechanism that is PAF independent.

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites.

Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.

The Agonist Binding Site on the Bovine Bradykinin B2 Receptor Is Adjacent to a Sulfhydryl and Is Differentiated from the Antagonist Binding Site by Chemical Cross-linking

Chemical cross-linking was used to analyze the binding sites for the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [3H]BK and [3H]NPC17731 bound with high affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific binding sites and pharmacological specificity of the binding of these two radioligands. Cross-linking experiments were done using a series of bifunctional reagents reactive either primarily to amines (homobifunctional) or reactive to amines in one end and to sulfhydryls in the opposite end (heterobifunctional). All the heterobifunctional reagents plus the homobifunctional arylhalide 1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the [3H]BK N terminus specifically to a sulfhydryl in the receptor, and this cross-linking occurred at 5-100 microM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at < or = 1 mM, were only able to cross-link [3H]BK to membrane proteins nonspecifically. The sulfhydryl reagents N-ethylmaleimide, iodoacetamide, and phenylarsine oxide blocked cross-linking, whereas these reagents did not inhibit reversible specific [3H]BK binding. Immunoblotting with anti-BK antiserum revealed that low concentrations of BK (5-50 nM) were cross-linked to a receptor-specific species of 65 kDa. All cross-linking of [3H]NPC17731 was nonspecific with both homobifunctional and heterobifunctional reagents. The 65-kDa receptor-specific species was observed on anti-HOE140 immunoblots, but only when proteins were cross-linked with very high concentrations of HOE140 (> or = 500 nM). Our results provide direct biochemical evidence that the binding site for the agonist BK in the bovine B2 receptor is adjacent to a cysteine and is differentiated from the binding site(s) for the antagonists NPC17731 and HOE140.

Strategy of Measuring Bradykinin and Kallidin and Their Concentration in Plasma and Urine

Bradykinin (BK) and kallidin (KAL) derivatives containing a Cys residue instead of a Ser residue at positions 6 and 7, respectively [BK(Cys6), KAL(Cys7)], were synthesized. These derivatives were linked to BSA via the Cys residue by a heterobifunctional cross-linker. The coupling product containing a kinin with both free N- and C-terminal ends was used as immunogen. We obtained highly sensitive and specific antisera, simultaneously directed against both free ends. The radioimmunoassay for BK displays a sensitivity of 0.5-60 fmol BK at a dilution of 1:80,000 with 125I-BK(Tyr8) as tracer. Des-Arg9-BK, [BK(1-8)], displayed the highest cross-reactivity in the amount of 24%. Des-Arg1-BK and smaller molecular weight fragments display a crossreactivity of less than 0.1%. The cross-reactivity of the BK antiserum with KAL is approximately 4%. In presence of 125I-KAL(Tyr9) the radioimmunoassay for KAL displays a sensitivity of 2 to 200 fmol KAL at an antiserum dilution of 1:80,000. The cross-reactivity with BK is 0.02%. KAL(Hyp4), BK(Hyp3), and des-Arg10-KAL [KAL(1-9)] show a cross-reactivity of 6.3, 4.9, and 2.4%. All other natural kinin derivatives show a cross-reactivity of less than 1%. Both assays were used to measure BK and KAL concentrations in blood and urine of humans after extraction and HPLC separation. The BK plasma level is 1.97 (SD 0.54) pg/ml. The KAL plasma level is 81.0 (SD 14.3) pg/ml, indicating that KAL instead of BK is a circulating peptide. In urine, the BK level is 16.3 pg/ml. As in plasma these values were much lower than those of urinary KAL (2120 pg/ml). These data suggest that kallidin may be either filtered or generated in the kidney, namely in the connecting tubuli, where high amounts of tissue kallikrein are known to be present. Furthermore, it appears that the small amounts of BK in urine are generated by conversion of KAL by urinary aminopeptidase B. In conclusion, highly selective antisera against BK and KAL were raised against both peptides coupled in the center of the molecule. These antibodies display a binding that is simultaneously directed against both free ends. With the radioimmunoassays for both peptides, high concentrations of KAL and low concentrations of BK are found in plasma and urine.

Plasma exudation in conscious dogs with experimental heart failure.

The purpose of this study was to determine the effects of bradykinin (BK), substance P (SP) and histamine on plasma exudation in the skin of conscious dogs with and without pacing-induced heart failure. We also determined the role tissue angiotensin I-converting enzyme (ACE) and neutral endopeptidase (NEP) play in modulating these responses. We found that intradermal injection of BK, SP and histamine induced a significant, concentration-dependent Evans blue exudation in normal dogs (p < 0.05). Bradykinin-induced responses were significantly potentiated by captopril (p < 0.05). In contrast, phosphoramidon potentiated BK-induced responses only at low concentrations of BK. Both captopril and phosphoramidon had no significant effects on SP- and histamine-induced Evans blue exudation. BK- and SP-induced responses were significantly attenuated, whereas histamine-induced Evans blue exudation was significantly potentiated in dogs with heart failure. We conclude that heart failure is associated with attenuation of BK- and SP-, but not histamine-induced plasma exudation in the peripheral microcirculation and that these responses are not modulated by tissue ACE and NEP.

Mechanism of [Ca2+]i oscillations in rat chromaffin cells. Complex Ca(2+)-dependent regulation of a ryanodine-insensitive oscillator

In the population of primary cultured rat chromaffin cells, over half exhibited spontaneous [Ca2+]i oscillations, whereas most others were induced to oscillate by low concentrations of bradykinin or KCl. [Ca2+]i spots were observed to pulsate in a defined cytoplasmic area (the oscillator). In silent cells those spots remained discrete, whereas in oscillating cells the [Ca2+]i increase expanded to occupy the entire cytoplasm. Alternation of these discrete and expanded events was observed in a few irregularly oscillating cells. Thapsigargin induced prompt blockade of both pulsations and oscillations and prevented recruitment of silent cells to oscillate. This indicates sarcoendoplasmic reticulum Ca(2+)-ATPase-type Ca2+ pump(s) to be crucial for the functioning of the oscillator. Effects of other treatments were variable, depending on the concomitant [Ca2+]i changes. Oscillations were blocked when EGTA or nitrendipine decreased Ca2+ influx and thus [Ca2+]i; they were also blocked when [Ca2+]i was markedly increased by excess KCl, bradykinin, or ryanodine. When in contrast the [Ca2+]i increases induced by the latter agents remained moderate, oscillations were stimulated. The rhythmic activity of rat chromaffin cells appears, therefore, to operate under a complex regulation that requires [Ca2+]i within an appropriate operative range and does not involve directly the ryanodine receptor but might rely on the activation of IP3 receptors.

Pharmacological characterization of the effects of 5-hydroxytryptamine and different prostaglandins on peripheral sensory neurons in vitro.

The excitatory and sensitizing properties of 5-HT and different prostaglandins (PGD2, PGE1, PGE2, PGF2 alpha, PGI2 and PGI2-analogue, cicaprost) were characterized on an in vitro preparation of the neonatal rat spinal cord with functionally attached tail. Prolonged (10 min) perfusion of the tail with 5-hydroxy-tryptamine (5-HT, 0.5-10 microM) or any of the tested prostaglandins (0.1-5 microM) did not evoke an excitatory response recorded from a lumbar ventral root, but significantly enhanced responses of peripheral nociceptors to thermal and chemical (bradykinin, capsaicin) stimuli. PGD2 did not induce such an enhancement. Following sensitization of peripheral nociceptors with low concentrations of bradykinin or capsaicin, 5-HT (1-10 microM) evoked a ventral root response. Using specific 5-HT-receptor agonists and antagonists, 5-HT-evoked excitation was determined to be mediated via a 5-HT1-like receptor while 5-HT-induced sensitization involved 5-HT2 receptors.

Sensitizing effects of bradykinin on the heat responses of the visceral nociceptor.

1. Whether bradykinin (BK), which is known as an endogenous pain-producing substance, induces augmentation of the discharges of polymodal receptors evoked by heat stimulation was investigated in in vitro canine testis-superior spermatic nerve preparations. 2. The heat response was significantly augmented by pretreatments with BK at concentrations greater than 0.094 nM, whereas BK induced significant increases in the mean discharge rates at concentrations above 9.4 nM. Both effects increased in a concentration-dependent manner. The augmenting effect of BK on the heat response diminished within 10 min after application of BK, regardless of concentration. 3. When 9.4 nM BK was applied in a mixture with 940 nM NPC349, a B2 receptor antagonist, the averaged mean discharge rate evoked by BK and the averaged augmenting effect were both significantly suppressed compared with those induced by BK given alone. 4. The augmenting effect of BK on the heat response of polymodal receptors could be observed even in the absence of BK-evoked discharges per se in several cases in which low concentrations of BK or BK plus B2 antagonists were given. 5. These findings suggest that the augmenting effects of BK on the heat response depend on B2 receptor-mediated intracellular processes acting in parallel to, but not directly on, the impulse-generation mechanism of the heat response of the polymodal receptor.

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP.

1. In bovine aortic endothelial cells (BAEC), thrombin (1 mu ml-1), bradykinin (1-10 nM) and adenosine triphosphate (ATP) (0.3 microM-100 microM) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. 2. Pretreatment of BAEC with 4 beta-phorbol 12-myristate 13-acetate (PMA; 100 nM) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 mu ml-1), low concentrations of bradykinin (1 nM) or ATP (0.3 microM, 3 microM), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nM) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (10 nM) or ATP (30 microM) resulted in a fall in [Ca2+]i. 3. The inhibitory effects of PMA (100 nM) were inhibited by staurosporine (100 nM) but not mimicked by the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD; 100 nM). Furthermore, staurosporine (100 nM) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nM) reduced [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 microM). 4. Pretreatment with forskolin (10 microM) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (1 nM and 10 nM) or ATP (30 microM). In contrast, forskolin (10 microM) and isoprenaline (10 microM) each induced biphasic elevations of [Ca21]i when added during the plateau phase of the increase in [Ca2+]i induced by the three agonists. Furthermore, in the presence of the inhibitor of calcium influx, nickel chloride (4mM), these biphasic elevations were reduced to monophasic transient elevations. 5. 8 Bromo cyclic GMP (30 microM), a membrane-permeant analogue of guanosine 3': 5'-cyclic monophosphate (cyclic GMP), had no effect on the magnitude of the initial transient elevation of [Ca21]i induced by thrombin (1 u ml 1), bradykinin (10 nM) or ATP (3 microM). Furthermore, 8 bromo cyclic GMP (30 microM) and sodium nitroprusside (1 microM), had no effect when added during the plateau phase of the increase in [Ca2+]i induced by the three agonists. 6. NG nitro-L-arginine (50,microM), an inhibitor of nitric oxide synthase, had no effect on the magnitude of the initial transient elevation of [Ca21]i induced by thrombin (1 uml- ), bradykinin (1 nM) or ATP (3,microM), and had no effect on the plateau phase of the increase in [Ca2+]i induced by these agents. 7. These findings suggest that while activation of protein kinase C inhibits and elevation of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) augments calcium mobilisation in bovine aortic endothelial cells, elevation of cyclic GMP appears to have no effect.

Ethanol effects on bradykinin-stimulated phosphoinositide hydrolysis in NG 108-15 neuroblastoma-glioma cells

The effect of short- and long-term ethanol exposure on bradykinin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2) was investigated in neuroblastoma × glioma hybrid cells (NG 108-15). Acute exposure of 50–150 mM ethanol neither influenced the bradykinin-stimulated accumulation of [ 3H]-inositol phosphates (IP 1, IP 2, IP 3) nor the hydrolysis of PIP 2 in cells labelled with [ 3H]-inositol. Furthermore, ethanol (100 mM) added in the absence of agonist did not influence these parameters. However, in cells cultivated for 4 days in 100 mM ethanol, PIP 2 hydrolysis and IP 1, IP 2 and IP 3 formation after stimulation by 10 −6–10 −5 M bradykinin was markedly inhibited while there was no effect on the basal levels or on the levels found after stimulation with low concentrations of bradykinin. The inhibitory effect of ethanol on IP accumulation became significant after 2–3 days of ethanol.

Effect of in vitro administration of captopril on vascular reactivity of rat aorta.

The effect of acute administration of captopril, an angiotensin converting enzyme inhibitor, on vascular responses of rings of rat aortic smooth muscle was tested in vitro. Dose-response curves for various vasoactive agents were obtained before and after exposure to captopril (2 x 10(-4) M) for 30 minutes. In the presence of captopril, contractile responses to angiotensin I (5 x 10(-10) to 5 x 10(-8) M) were attenuated significantly, probably as a result of decreased local conversion of angiotensin I to angiotensin II. Contractile responses to angiotensin II (10(-11) to 5 x 10(-9) M) were not affected by captopril. All responses to norepinephrine (10(-9) to 10(-4) M) and phenylephrine (10(-8) to 10(-4) M) were attenuated significantly from control in the presence of captopril. In the presence of the alpha-adrenergic antagonist, phentolamine, captopril did not affect either the contractile responses to KCl (30 to 100 mM) or the isoproterenol-induced (10(-8) to 10(-5) M) relaxation of KCl-depolarized tissue. These results suggest that captopril decreased vascular responsiveness to alpha-adrenergic agonists but not to beta-adrenergic agonists. Low concentrations of bradykinin (10(-10) to 10(-8) M) induced contraction in KCl-depolarized tissue while higher concentrations (10(-7) and 10(-6) M) induced relaxation. In the presence of captopril, relaxation occurred at all concentrations of bradykinin (10(-10) to 10(-6) M), probably as a result of decreased degradation of the bradykinin. These data suggest depression of alpha-adrenergic responsiveness in vascular smooth muscle as another potential antihypertensive action of captopril.