Bradykinin-induced Prostaglandin Research Articles

Resveratrol prevents bradykinin-induced contraction of rat urinary bladders by decreasing prostaglandin production and calcium influx

Resveratrol, a polyphenol found in grapes and peanuts, exerts beneficial effects on a number of diseases of cardiovascular and central nervous system. However, effects of resveratrol on the urinary system have not been fully investigated. In the present study, we examined effects of resveratrol on bradykinin-induced contraction and release of prostaglandin E 2 in isolated rat urinary bladders. The effects of resveratrol on contractions induced by several agonists (prostaglandin E 2, prostaglandin F 2α and carbachol) and high K + were also examined. We found that resveratrol concentration-dependently reduced the bradykinin-induced contraction in the rat urinary bladder preparations. The higher concentration of resveratrol (100 μM) abolished the bradykinin-induced prostaglandin E 2 release. Similar results were obtained when the cyclooxygenase inhibitor indomethacin (10 μM) was used instead of resveratrol. Resveratrol also attenuated the prostaglandin E 2-, prostaglandin F 2α-, and to a lesser extent carbachol-induced contractions. Contractile responses to bradykinin, prostaglandin E 2 and carbachol were largely prevented by blockade of Ca 2+ channels with diltiazem. Both resveratrol and diltiazem prevented contractions induced by an addition of Ca 2+ (2.5–10 mM) into Ca 2+-free/50 mM K + solution or by 50 mM K + solution containing normal Ca 2+ (2.5 mM). These results suggest that resveratrol prevents bradykinin-induced contractions by attenuating not only the production of prostaglandins but also actions of them. The effect of resveratrol on contractile actions seems to be in part due to inhibition of Ca 2+ influx. Because bradykinin plays an important role in pathological conditions of urinary bladder function, resveratrol may exert beneficial effects on the urinary bladder diseases.

Alleviating Effects of AS1892802, a Rho Kinase Inhibitor, on Osteoarthritic Disorders in Rodents

The effects of AS1892802, a selective Rho-associated coiled coil kinase (ROCK) inhibitor, on knee cartilage damage and pain behavior were examined in a rat model of osteoarthritis (OA). Monoiodoacetate (MIA) was intraarticularly injected into the right knee joints of rats. ROCK I and II mRNA levels increased in knee joints of MIA-injected rats. Our newly synthesized ROCK inhibitor, AS1892802, was injected into the ipsilateral knee or administered p.o. for 3 weeks. The compound dose-dependently and significantly inhibited of cartilage damage in the tibial plateau in a dose-dependent manner and decreased the weight distribution deficit associated with MIA injection. In addition, the compound also inhibited bradykinin induced pain responses in normal rats. In vitro, the compound could induce chondrocyte differentiation in a chondrogenic cell line and significantly inhibited IL-1β– or bradykinin-induced prostaglandin E2 production in a synovial cell line. AS1892802 prevents cartilage damage induced by MIA and has analgesic effects in rat pain models, suggesting that AS1892802 may be clinically useful for the treatment of OA.[Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.10319FP]

Regulation of glial cyclooxygenase by bradykinin

The aim of the present study was to investigate the short-term effect of bradykinin on the two cyclooxygenase species in neonatal rat glial cells. In spite of the fact that cyclooxygenase protein levels were not altered, an increase in cyclooxygenase activity was observed. Use of cyclooxygenase-1 inhibitors and paracetamol resulted in complete elimination of the bradykinin-induced prostaglandin E 2 synthesis and of cyclooxygenase enzyme activity. Cyclooxygenase-2 inhibitors only partially inhibited enzyme activity and prostaglandin production. Our data suggest that cyclooxygenase-1 is probably the major contributor to short-term modulation of glial prostaglandin E2 synthesis by bradykinin.

Dietary Lutein/Zeaxanthin Decreases Ultraviolet B-Induced Epidermal Hyperproliferation and Acute Inflammation in Hairless Mice

Lutein and zeaxanthin are carotenoids found in green leafy vegetables with interesting antioxidant properties. They are present in high concentrations in the fovea centralis of the human retina and their role in the prevention of age-related macula degeneration has been reported. We have investigated the effect of orally administered lutein and zeaxanthin in the cutaneous response to ultraviolet B irradiation. Female hairless SKh-1 mice receiving 0.4% and 0.04% lutein plus zeaxanthin-enriched diet for 2 wk were exposed to single doses of ultraviolet B radiation. Skin biopsies were taken at 24 and 48 h after irradiation and analyzed for the presence of apoptotic cells, proliferating cells, and expression of proliferating cell nuclear antigen. Our results show a clear ultraviolet-induced dose-dependent inflammatory response. Orally administered 0.4% lutein and zeaxanthin decreased significantly the edematous cutaneous response (p<0.01) as determined by the reduction of the UVB-induced increase of ear bifold thickening. Additionally, dietary carotenoids were efficient in reducing the ultraviolet B-induced increases in the percentage of proliferating cell nuclear antigen (p<0.05), bromodeoxyuridine (p<0.05), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells (p<0.01). These data demonstrate that oral supplementation of lutein and zeaxanthin diminishes the effects of ultraviolet B irradiation by reducing acute inflammatory responses and ultraviolet-induced hyperproliferative rebound.

Characterization of bradykinin-induced prostaglandin E 2 release from cultured rat trigeminal ganglion neurones

Bradykinin and prostaglandins are both local mediators strongly implicated in pain and inflammation. Here, we have investigated the effects of bradykinin on the release of prostaglandin E 2 from cultured neurones derived from adult rat trigeminal ganglia. Bradykinin was a potent inducer of prostaglandin E 2 release, an effect that was likely mediated by bradykinin B 2 receptors, as the bradykinin-induced prostaglandin E 2 release was attenuated by the bradykinin B 2 receptor-selective antagonist, arginyl- l-prolyl-trans-4-hydroxy- l-prolylglycyl-3-(2-thienyl)- l-alanyl- l-seryl- d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl- l-(2α, 3β, 7aβ)-octahydro-1 H-indole-2-carbonyl- l-arginine (HOE 140), but not by the bradykinin B 1 receptor-selective antagonist, des-Arg 9,[Leu 8]-bradykinin. Furthermore, bradykinin-induced prostaglandin E 2 release was inhibited following treatment with the phospholipase A 2 inhibitor, arachidonyltrifluoromethyl ketone (AACOCF 3), the nonselective cyclooxygenase inhibitor, piroxicam, the mitogen-activated protein kinase kinase-1 (MEK1) inhibitor, 2′-amino-3′-methoxyflavone (PD98059), and the protein kinase C inhibitor, bisindolylmaleimide XI (Ro320432). Taken together, these data suggest that bradykinin, acting via bradykinin B 2 receptors, induces prostaglandin E 2 release from trigeminal neurones through the protein kinase C and mitogen-activated protein kinase-dependent activation of phospholipase A 2 and consequent stimulation of cyclooxygenases.

Preconditioning of 3T3 cells by fresh medium together with genistein enhances prostaglandin E 2 release

Bradykinin induced prostaglandin E 2 release from the Swiss 3T3 fibroblasts, preconditioned with fresh culture medium. Although treatment with genistein for the entire period of preconditioning and incubation with bradykinin attenuated prostaglandin E 2 release, treatment with fresh culture medium and genistein for only the preconditioning period further augmented the prostaglandin E 2 release. In the cells preconditioned with fresh culture medium and genistein, bradykinin caused the phosphorylation of protein tyrosine and mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK), followed by arachidonic acid release. Interestingly, preconditioning with genistein alone also caused phosphorylation and arachidonic acid release, probably reflecting rebound activation after the washout of genistein. However, preconditioning with genistein alone induced neither the augmentation of prostaglandin E 2 release nor the expression of cyclooxygenase-2. The further potentiation of bradykinin-induced prostaglandin E 2 release by combined preconditioning with fresh culture medium and genistein may be due to the activation of the MAPK/ERK-c phospholipase A 2 pathway by preconditioning with genistein.

Bradykinin induces a rapid cyclooxygenase-2 mRNA expression via Ca2+mobilization in human gingival fibroblasts primed with interleukin-1 β

We have previously demonstrated that bradykinin potentiates prostaglandin E2release in human gingival fibroblasts pretreated with interleukin-1β (priming). In this study, we demonstrate a potentiating effect of bradykinin on cyclooxygenase-2 mRNA expression in the interleukin-1β-primed fibroblasts. Interleukin-1β (200pg/ml) induced cyclooxygenase-2 mRNA expression, but not bradykinin (1μM). However, bradykinin rapidly and markedly increased the cyclooxygenase-2 mRNA expression in the fibroblasts primed with interleukin-1β. In the primed fibroblasts, ionomycin and thapsigargin mimicked the potentiating effect of bradykinin on the cyclooxygenase-2 mRNA expression. Dexamethasone and actinomycin D completely suppressed not only the interleukin-1β-induced cyclooxygenase-2 mRNA expression, but also the bradykinin-induced cyclooxygenase-2 mRNA expression in the interleukin-1β-primed fibroblasts, although cycloheximide did not inhibit the effects of interleukin-1β and bradykinin. These results suggest that bradykinin-induced prostaglandin E2synthesis is regulated at the level of the transcription of cyclooxygenase-2 mRNA via Ca2+mobilization in the interleukin-1β-primed human gingival fibroblasts.

Altered coronary dilation in deoxycorticosterone acetate-salt hypertension.

To compare coronary dilation in uninephrectomized hypertensive deoxycorticosterone acetate (DOCA)-salt rats (HTRs), treated for 2 or 4 weeks, with age-matched uninephrectomized normotensive rats (NTRs). DESIGN AND METHODS Coronary perfusion pressure was recorded in isolated hearts perfused at a constant flow rate to evaluate coronary resistance. A decreased vasoconstriction due to NG-nitro-Larginine (NNLA, 30 pmol/I) in hearts from HTRs suggested a reduced basal nitric oxide (NO) release. In contrast, coronary vasodilation due to the NO donor, sodium nitroprusside (3 pmol/I), remained unaffected in 2-week HTRs, and was enhanced in 4-week HTRs. Cumulative dose-response curves to bradykinin induced an important vasodilation in NTRs, with a maximal response that remained unaffected in the presence of either NNLA (30 pmol/I), indomethacin (10 pmol/l) or the two combined. In contrast, hearts from HTRs showed a diminished maximal relaxation to bradykinin, suggesting an altered endothelium-dependent relaxation. The presence of NNLA or indomethacin had no effect on the weak relaxation observed in HTRs. However, NNLA and indomethacin combined unmasked an important relaxation due to bradykinin in HTRs. The addition of clotrimazole (1 pmol/I) to NNLA and indomethacin blunted the relaxation due to bradykinin in both NTRs and HTRs. Perfusion with superoxide dismutase (120 IU/ml) restored most of the coronary relaxation due to bradykinin in hearts from HTRs. Bradykinin-induced prostaglandin 12 (PGI2) and E2 (PGE2) production was unaffected by hypertension. No increase in thromboxane A2 (TXA2) due to bradykinin was detected. Finally, reduced reactivity to papaverine and forskolin was observed in hearts from HTRs. DOCA-salt hypertension is associated with alterations in coronary reactivity. Basal NO formation appears to be reduced in HTRs, but the intact relaxation to exogenous NO suggests a preserved guanylate cyclase pathway. In addition, alteration in adenylate cyclase activity, and not in prostaglandin production, may explain the blunted cAMP-mediated responses in HTRs. The combined nitric-oxide synthase (NOS) and cyclo-oxygenase (COX) inhibition unmasked an endothelium-derived hyperpolarizing factor (EDHF) involvement in the coronary dilation due to bradykinin in hearts from HTRs, suggesting that endothelial NO and PGI2, although unable to induce coronary smooth-muscle relaxation, can inhibit EDHF production in HTRs. Impairment in the adenylate cyclase pathway and the suppression of NO by free radicals may explain the blunted vasodilation in DOCA-salt hypertension.

Involvement of the endogenous nitric oxide signalling system in bradykinin receptor activation in rat submandibular salivary gland

Biochemical signalling events coupled to the bradykinin B 2-receptor subtype, related to nitric oxide and prostaglandin E 2 generation were studied in rat submandibular gland. Bradykinin stimulation of the B 2-receptor triggered activation of phosphoinositide turnover, translocation of protein kinase C, stimulation of nitric oxide synthase activity, increased production of cGMP and release of prostaglandin E 2. Bradykinin stimulation of nitric oxide synthase and cGMP production was blunted by agents able to interfere with calcium/calmodulin and phospholipase C activities, while a protein kinase C inhibitor was able to stimulate bradykinin action. Moreover, a specific B 2-bradykinin antagonist of the reversible nitric oxide synthase inhibitor abrogated the bradykinin stimulation of nitric oxide synthase activity, cGMP accumulation and prostaglandin E 2 generation. Furthermore, a specific inhibitor of phospholipase A 2 blocked the bradykinin-induced prostaglandin E 2 release. These results suggest that apart, from the direct effect of bradykinin as an inducer of vasopermeability, it also appears to be a vasoactive chemical mediator that triggers, through release of prostaglandin E 2, a feedback mechanism that induces a protective adaptation of the gland, modulating the course of inflammation.

Bradykinin potentiates prostaglandin E 2 release in the human gingival fibroblasts pretreated with interleukin-1β via Ca 2+ mobilization

Interleukin-1β, a proinflammatory cytokine, causes a slow increase in prostaglandin E 2 release. On the other hand, bradykinin, a chemical mediator for inflammation, induces a rapid prostaglandin E 2 release. Simultaneous stimulation with interleukin-1β (200 pg/ml) and bradykinin (1 μM) evoked a moderately synergistic increase in prostaglandin E 2 release in human gingival fibroblasts. However, in the human gingival fibroblasts pretreated with interleukin-1β, bradykinin drastically enhanced prostaglandin E 2 release. NS-398, a specific inhibitor of cyclooxygenase-2, inhibited not only interleukin-1β-induced prostaglandin E 2 release but also bradykinin-induced prostaglandin E 2 release in the human gingival fibroblasts pretreated with interleukin-1β. Transcriptional and translational inhibitors such as actinomycin D, cycloheximide, and dexamethasone also suppressed the interleukin-1β-induced prostaglandin E 2 release and the bradykinin-induced prostaglandin E 2 release in interleukin-1β-pretreated human gingival fibroblasts. In the fibroblasts pretreated with interleukin-1β, Ca 2+-mobilizing reagents such as ionomycin and thapsigargin mimicked the potentiating effect of bradykinin on prostaglandin E 2 release. These results suggest that interleukin-1β- and bradykinin-induced prostaglandin E 2 release is dependent on cyclooxygenase-2 and the potentiated effect of bradykinin in the human gingival fibroblasts primed with interleukin-1β is caused by Ca 2+ mobilization.

Medium change amplifies mitogen-activated protein kinase-mediated prostaglandin E 2 synthesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts, changing the culture medium prior to stimulation resulted in an augmentation of bradykinin-induced prostaglandin E 2 synthesis. The augmentation depended on the duration of the exposure to the fresh medium, with a maximum effect at 1 h. Fetal calf serum in the fresh medium was essential for augmented prostaglandin E 2 synthesis. The medium change slightly augmented the bradykinin-induced increase in intracellular free Ca 2+ concentration and phosphoinositide hydrolysis with a different time course from that for prostaglandin E 2 synthesis. 4′,5,7-Trihydroxyisoflavone (genistein) and 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitors of tyrosine kinases, and 2′-amino-3′-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase (MAPK) kinase, attenuated the increase in prostaglandin E 2 synthesis. Bradykinin caused phosphorylation of cytosolic phospholipase A 2 and p42/p44 MAPK, which was augmented by the medium change. From the results, it is concluded that activation of MAPK and cytosolic phospholipase A 2 is involved in the augmentation of prostaglandin E 2 synthesis produced by the medium change.

Inhibition of prostanoid formation in intact cells by 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a blocker of Ca(2+)-ATPases.

1 The blocker of endoplasmic reticulum Ca(2+)-ATPase, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast-like cell lines, MC3T3-E1 and ROS 17/2.8, respectively, in a dose-dependent manner with an IC50 of 0.5-1 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187). 2 This effect was also observed in human platelets, where BHQ dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12-hydroxy-eicosatetraenoic acid. 3 Inhibition of prostaglandin E2 formation in MC3T3-E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin-induced prostaglandin E2 biosynthesis was blocked. 4 Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo-oxygenase in these three cell systems.

In vivo and in vitro homologous desensitization of rat glomerular bradykinin B 2 receptors

We investigated the effects of bradykinin on glomerular bradykinin B 2 receptor functions and parameters in vivo, after intrarenal infusion of bradykinin, and in vitro, after incubation of isolated rat glomeruli with bradykinin. Bradykinin transiently increased renal plasma flow whereas a second challenge was ineffective. Scatchard analysis demonstrated the presence of two populations of bradykinin binding sites whose densities were similarly decreased by about 40% after intrarenal bradykinin infusion. This decrease was not altered by an acid wash suggesting internalization of the radiolabelled ligand. The effect of bradykinin was prevented by a bradykinin B 2 receptor antagonist. Pre-exposure of isolated rat glomeruli to bradykinin mimicked the in vivo results because there was a reduction in bradykinin-induced prostaglandin E 2 and prostagandin F 2a release. Rapid recovery was observed 15 min after washing out the bradykinin. Our results directly demonstrate a negative homologous down-regulation of B 2 glomerular bradykinin receptor density under both in vivo and in vitro conditions, an effect which involves a rapid sequestration of the receptor.

Relationship of Arachidonic Acid Release to Porcine Coronary Artery Relaxation

In porcine coronary artery endothelium-dependent relaxation to bradykinin is in part attributed to a chemically unidentified factor, termed endothelium-derived hyperpolarizing factor (EDHF). We hypothesize that arachidonic acid, acting through a cyclooxygenase-independent mechanism, is responsible for EDHF production. To define the relationship between EDHF production and arachidonic acid release, we investigated the role of phospholipase C in bradykinin-induced relaxation and prostaglandin I2 production (an index of arachidonic acid release) in porcine coronary artery. The phospholipase C inhibitor U73122 (1 mumol/L) abolished bradykinin-induced, nitric oxide-mediated relaxation but did not inhibit either bradykinin-induced, EDHF-mediated relaxation or prostaglandin I2 production. However, when given at a larger dose (20 mumol/L) U73122 abolished both bradykinin-induced, EDHF-mediated relaxation and prostaglandin I2 production. Similarly, the calcium-ATPase inhibitor thapsigargin, given at a dose (1 mumol/L) that abolished bradykinin-induced increases in intracellular calcium concentration in cultured porcine coronary artery endothelial cells, eliminated both bradykinin-induced. EDHF-mediated relaxation and prostaglandin I2 production. Although thapsigargin abolished bradykinin-induced prostaglandin I2 production, the basal production of prostaglandin I2 was enhanced and contraction of endothelium-intact rings was attenuated. These latter responses are most likely related to enhanced basal arachidonic acid release and associated EDHF production. These observations suggest that phospholipase C activation and increased intracellular calcium concentration are required for both bradykinin-induced arachidonic acid release and EDHF production in porcine coronary artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [ 3H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg 9-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE 2) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca 2+ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE 2 formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE 2 formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.

Bradykinin-induced prostaglandin synthesis is enhanced in keratinocytes and fibroblasts by UV injury

Ultraviolet (UV) light exposure substantially modifies the host immune response, in part through the synthesis of prostaglandins. Work examining the mechanisms by which UV irradiation stimulates prostaglandin synthesis has focused on mediators that increase in quantity after irradiation. The present work demonstrates that UV irradiation injury increases the sensitivity and maximum response of keratinocytes to bradykinin, suggesting that agonist quantities need not increase in injured tissue to contribute to inflammation. The ability of UV injury to increase bradykinin-stimulated prostaglandin synthesis is not limited to keratinocytes, as irradiation produced a similar response in fibroblasts. Receptor binding studies demonstrate that the enhanced response of irradiated cells is not due to increased bradykinin binding. These data suggest that UV irradiation may cause cells to increase their response to an array of inflammatory mediators present in injured tissue, whether or not the quantity of the mediator increases.

Evidence that Cultured Airway Smooth Muscle Cells Contain Bradykinin B2 and B3 Receptors

We examined bradykinin-induced 45Ca2+ efflux and prostaglandin synthesis in guinea pig tracheal smooth muscle cells maintained in tissue culture. We also studied the effects of a B1 receptor agonist and antagonist, a B2 receptor antagonist, and the cyclooxygenase inhibitor indomethacin. In cultured smooth muscle cells, bradykinin (0.1 nM to 10 microM) stimulated efflux of 45Ca2+ and induced the synthesis of prostaglandin E2 and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha. DesArg9-bradykinin, a B1 receptor agonist, had no effect on 45Ca2+ efflux or prostaglandin synthesis, and no responses to bradykinin were unaffected by the B1 receptor antagonist desArg9-[Leu8]-bradykinin. Indomethacin (1 microM) abolished bradykinin-induced prostaglandin synthesis but was without effect on 45Ca2+ efflux. NPC 567 (DArg[Hyp3,DPhe7]-bradykinin), a B2 receptor antagonist, had no effect on bradykinin-induced 45Ca2+ efflux, but abolished prostaglandin synthesis. Unlike in membranes prepared freshly from guinea pig tracheal smooth muscle, the B2 receptor antagonist inhibited completely (Ki, 12 nM) binding of [3H]-bradykinin to membranes prepared from cultured tracheal smooth cells. These data suggest that tracheal smooth muscle cells, maintained in culture, express B2 receptors that mediate bradykinin-induced prostaglandin synthesis. The observation that bradykinin-induced efflux of calcium ions was unaffected by B1 or B2 antagonists provides further evidence that airway smooth muscle may contain a novel B3 receptor.

Enhancement of bradykinin-induced prostacyclin synthesis in porcine aortic endothelial cells by pertussis toxin: Possible implication of lipocortin I

Bradykinin-stimulated prostacyclin synthesis in porcine aortic endothelial cells was enhanced by pretreatment of the cells with pertussis toxin or islet-activating protein (IAP) for 5 hr or longer. Although ADP-ribosylation of a protein with a molecular weight of 41–42 kD in the cell membranes was completed by 3 hr after the addition of IAP into the incubation medium, there was good correlation between enhancement of bradykinin-induced prostacyclin synthesis and ADP-ribosylation of the IAP substrate over a wide range of IAP concentrations. Furthermore, even if IAP was removed from the incubation medium at 3 hr, bradykinin-induced prostaglandin synthesis at 24 hr was still potentiated. Cycloheximide and actinomycin D enhanced bradykinin-induced prostacyclin synthesis and apparently blocked the effect of IAP. Since this result suggested the involvement of an inhibitor protein(s) of prostacyclin synthesis in the IAP effect, we studied the effect of IAP on the level of lipocortin I which is known to inhibit phospholipase A 2. Western and Northern blot analyses revealed that IAP decreased the amounts of protein and mRNA of lipocortin I. These results suggest that the enhancement of bradykinin-induced prostacyclin synthesis by IAP is associated with a decrease in the level of lipocortin I.

Bradykinin stimulates production of prostaglandin E 2 and prostacyclin in murine osteoblasts

The effect of bradykinin on prostaglandin production in mouse calvarial bones and in isolated osteoblasts has been examined. Bradykinin (1 μmol/l) stimulated prostaglandin formation in neonatal mouse calvarial bones incubated for 30 min. In isolated osteoblast-like cells from neonatal mice calvarial bones and in a cloned mouse calvarial osteoblastic cell lineage (MC3T3-E1) bradykinin stimulated the production of prostaglandin E 2 (PGE 2) and 6-keto-prostaglandin F 1α (the stable breakdown product of prostacyclin). The stimulation of PGE 2 production occurred rapidly (30 s) and reached its maximum after 5–10 min. The stimulatory effect of bradykinin on PGE 2 production in isolated osteoblast-like cells and in MC3T3-E1 cells was dose dependent with apparent half maximal stimulation seen at 10 and 3 nmol/l, respectively. Bradykinin-induced prostaglandin production was totally reversible after withdrawal of the agonist. Pretreatment with bradykinin (1 μmol/l) resulted in desensitization to a subsequent challenge with bradykinin (1 μmol/l), while pretreatment with bradykinin had no effect upon arachidonic acid (30 μmol/l) induced prostaglandin formation. Bradykinin-induced production of PGE 2 was abolished by several structurally unrelated, competitive and non-competitive inhibitors of arachidonic acid metabolism as well as by corticosteroids. The mouse calvarial osteoblast-like cells also showed a PGE 2 and 6-keto-PGF 1α response to thrombin, but not to parathyroid hormone (PTH), calcitonin and 1α(OH)D 3. The formation of cyclic AMP in mouse calvarial osteoblasts was enhanced by PTH, bradykinin, thrombin and arachidonic acid but not by calcitonin and 1α(OH)D 3. The cyclic AMP response to bradykinin, thrombin and arachidonic acid, but not that to PTH, was abolished by indomethacin. The degree of confluency of the cell cultures greatly influenced the amount of prostaglandins being produced. At higher cell density the amount of prostanoids synthesized per cell was substantially decreased in untreated control cultures as well as in bradykinin- and arachidonic acid-treated cells. These data suggest that osteoblasts are equipped with receptors for bradykinin coupled to prostaglandin production.

Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2.

In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E2 (PGE2) synthesis. The EC50 values for stimulation of PGE2 synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-[gamma-thio]triphosphate stimulated PGE2 synthesis and InsP formation, and guanosine-5'-[beta-thio]diphosphate inhibited both PGE2 synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE2 synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [3H]choline, the phospholipase A2 products lysophosphatidylcholine and glycerophosphocholine were generated. In cells pretreated with dexamethasone, lysophosphatidylcholine and glycerophosphocholine formation induced by bradykinin were inhibited. Treatment of cells with phorbol ester enhanced bradykinin-induced formation of these metabolites. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A2 and that phospholipase A2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.