Bradykinin induces a B2 receptor-mediated calcium signal linked to prostanoid formation in human gingival fibroblasts in vitro.

The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca2+, [Ca2+]i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg9-BK, induced a significant rapid (within seconds) and transient increase in [Ca2+]i, that was not dependent on extracellular Ca2+. The stimulatory effect of BK was seen in concentrations at or above 10(-8) M, with the most pronounced effect at 10(-6) M. D-Arg0-Hyp3-Thi5,8-DPhe7-BK, a BK B2 receptor antagonist, but not des-Arg9-Leu8-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca2+]i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE2 formation. When extracellular Ca2+ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca2+ addition, BK still caused a significant stimulation of PGE2 formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE2 formation. The two phorbol esters phorbol 12,13-dibutyrate and 4-beta-phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE2 formation. The results indicate that BK-induced PGE2 formation in gingival fibroblasts is coupled to an increase in [Ca2+]i mediated by the BK B2 receptor, and which is independent of extracellular Ca2+.     

Fasitibant prevents the bradykinin and interleukin 1β synergism on prostaglandin E2 release and cyclooxygenase 2 expression in human fibroblast-like synoviocytes

This study investigates the effect of the selective and potent B(2) receptor antagonist fasitibant (MEN16132) on the proinflammatory effect of bradykinin (BK) and its interaction with interleukin 1β (IL-1β) in human synoviocytes. PGE(2) content was detected in the surnatants and COX-2 and COX-1 gene and protein expression determined in the cells. Radioligand binding ([(3)?H]BK) and BK-induced inositolphosphate experiments were performed. Incubation of synoviocytes with BK induced a sustained production of PGE(2) and transient COX-2 gene expression that were prevented by pretreatment with fasitibant (1 μM, 30 min preincubation). IL-1β increased PGE(2) release and COX-2 expression more than BK alone. The combined treatment of cells with BK and IL-1β induced an even increase of released PGE(2) and COX-2 gene and protein expression indicating a synergistic rather than an additive effect, not related to an increase of B(2) receptors density or its coupling. These potentiating effects of BK on PGE(2) production and increased COX-2 expression produced by IL-1β were B(2)-receptor-mediated as fasitibant could prevent them. None of the treatments induced changes in the COX-1 expression. The synergistic PGE(2) production was abolished by the specific NF-kappaB inhibitor (BAY-117085), whereas specific inhibitors for the p38 (SB203580), JNK (SP600125), and ERK1/2 (PD98059) mitogen-activated protein kinases could prevent the prostanoid release. BK can potentiate the COX-2 gene expression and consequent prostanoid production induced by IL-1β. The prevention of this synergism by fasitibant indicates BK B(2) receptor blockade as an alternative symptomatic therapy for osteoarthritis.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells

1. Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). 2. BK and Lys-BK caused an initial transient peak of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. 3. Kinin B2 antagonists Hoe 140 (10 nM) and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (1 microM) had high affinity in antagonizing BK-induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. 4. Pretreatment of TECs with pertussis toxin (100 ng ml(-1)) or cholera toxin (10 microg ml(-1)) for 24 h did not affect the BK-induced IP accumulation and [Ca2+]i changes in TECs. 5. Removal of Ca2+ by the addition of EGTA or application of Ca2+-channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK-induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK-induced responses. 6. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+-free buffer and subsequently induced Ca2+ influx when Ca2+ was re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca2+]i, but had slight effect on BK-induced Ca2+ influx. 7. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor-gated Ca2+-channels and phospholipase C in TECs, respectively. 8. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C-mediated IP accumulation and Ca2+ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK-induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.     

Inositol 1,4,5-triphosphate-mediated shuttling between intracellular stores and the cytosol contributes to the sustained elevation in cytosolic calcium in FMLP-activated human neutrophils

The current study was designed to probe Ca2+ shuttling between intracellular stores and the cytosol as a potential mechanism contributing to the prolongation of elevated Ca2+ transients in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation were measured using spectrofluorimetric and radiometric procedures, respectively, while inositol 1,4,5-triphosphate (IP3) was measured using a radioreceptor assay. The Ca2+-chelating agent, ethylene glycol-bis (beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA; 10mM), was used to exclude store-operated influx of Ca2+ into neutrophils, while the IP3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100 microM), added to the cells 10s after FMLP (0.01 and 1 microM), at which time the increases in IP3 and cytosolic Ca2+ were maximal, was used to eliminate both sustained release from stores and influx of Ca2+. Addition of FMLP at 0.01 or 1 microM resulted in equivalent peak increases in cytosolic Ca2+, while the increase in IP3 was greater and the rate of clearance of Ca2+ from the cytosol slower, in cells activated with 1 microM FMLP. Treatment of the cells with either EGTA or 2-APB following addition of 1 microM FMLP, completely (EGTA) or almost completely (2-APB) abolished the influx of Ca2+ and accelerated the rate of clearance of the cation from the cytosol. Post-peak cytosolic Ca2+ concentrations were lower, and the Ca2+ content of the stores higher, in cells treated with 2-APB. The involvement of IP3 was confirmed by similar findings in cells treated with U-73122 (1 microM), a selective inhibitor of phospholipase C. Taken together, these observations are compatible with IP3-mediated Ca2+ shuttling in neutrophils activated with FMLP.     

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway

We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.     

Mechanisms underlying the relaxation response induced by bradykinin in the epithelium-intact guinea-pig trachea in vitro

In this study, we investigated some of the signalling pathways involved in bradykinin (BK)-induced relaxation in epithelium-intact strips of the guinea-pig trachea (GPT + E). BK induced time- and concentration-dependent relaxation of GPT + E. Similar responses were observed for prostaglandin E2 (PGE2) or the combination of subthreshold concentrations of BK plus PGE2. The nonselective cyclooxygenase (COX) inhibitors indomethacin or pyroxicam, or the selective COX-2 inhibitors DFU, NS 398 or rofecoxib, but not the selective COX-1 inhibitor SC 560, all abolished BK-induced relaxation. The tyrosine kinase inhibitors herbimycin A and AG 490 also abolished BK-induced relaxation in GPT + E. The nonselective nitric oxide synthase (NOS) inhibitor 7-NINA concentration-dependently inhibited BK effects. BK-induced relaxation was prevented by the selective antagonists for EP3 (L 826266), but not by EP1 (SC 19221), EP1/EP2 (AH 6809) or EP4 (L161982) receptor antagonists. Otherwise, the selective inhibitors of protein kinases A, G and C, mitogen-activated protein kinases, phospholipases C and A2, nuclear factor-kappaB or potassium channels all failed to significantly interfere with BK-mediated relaxation.BK caused a marked increase in PGE2 levels, an effect that was prevented by NS 398, HOE 140 or AG 490. COX-2 expression did not differ in preparations with or without epithelium, and it was not changed by BK stimulation. However, incubation with BK significantly increased the endothelial NOS (eNOS) and neuronal NOS (nNOS) expression, independent of the epithelium integrity. Our results indicate that BK-induced relaxation in GPT + E depends on prostanoids (probably PGE2 acting via EP3 receptors) and NO release and seems to involve complex interactions between kinin B2 receptors, COX-2, nNOS, eNOS and tyrosine kinases.     

Bradykinin stimulates production of inositol (1,4,5) trisphosphate in cultured mesangial cells of the rat via a BK2-kinin receptor.

1. Using [125I-Tyr0]-BK, as radiolabelled ligand, and various agonists and antagonists of bradykinin (BK) we identified a single class of specific BK2-binding sites in mesangial cell membranes (Bmax = 73 fmol mg-1 protein and Kd = 3.7 nM). 2. Following the addition of 0.1 microM BK, inositol (1,4,5) trisphosphate (IP3) formation increased within 20 s from a basal level of 64 to a maximal value of 175 pmol mg-1 protein. 3. Incubation in a Ca(2+)-free medium did not change IP3 production but a 5 min preincubation with 1 mM EGTA completely prevented the BK-induced IP3 formation, suggesting that IP3 formation is partly dependent on extracellular calcium. 4. The BK2 antagonist D-Arg-Hyp3-D-Phe7-BK (10 microM) but not the BK1 antagonist (des-Arg9-Leu8-BK) abolished IP3 production in response to 0.1 microM BK. Pretreatment of mesangial cells with pertussis toxin was without effect on BK-induced IP3 formation, whereas phorbol 12-myristate 13-acetate significantly enhanced (by 25%) BK-induced IP3 formation. 5. The present data demonstrate that inositol phosphate breakdown in rat mesangial cells can be mediated via activation of a BK2-kinin receptor and is under negative control of protein-kinase C.     

Bradykinin-induced cyclic AMP accumulation in mouse fibrosarcoma independent of prostaglandin E2 formation

The relationship between bradykinin (BK)-induced prostaglandin E2 (PGE2) and cyclic AMP syntheses in mouse fibrosarcoma cells (HSDM1C1) was investigated. Maximal BK-induced increases in cyclic AMP preceded increases in PGE2 production. PGE2 synthesis reached maximum at a much lower concentration of BK than cyclic AMP synthesis. Indomethacin completely inhibited BK-induced PGE2 production, but did not influence the cyclic AMP levels. Arachidonic acid in the medium induced PGE2 production in large quantities, but increased cyclic AMP accumulation only slightly. A high PGE2 concentration increased cyclic AMP levels only slightly. Theophylline increased basal and BK-mediated cyclic AMP levels, but did not affect PGE2 production at all. These results indicate that BK-evoked PGE2 and cyclic AMP syntheses in HSDM1C1 are not dependent upon each other.     

Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y1 receptor in a Ca2+ independent manner in differentiated human skeletal muscle cells

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca2+ transients in these cells via P2Y receptors which were not attributable to Ca2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC50 of 21.3 microM. The Ca2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca2+ pool was not involved in ATP triggered Ca2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Substantially, ATP activated IP formation via a P2Y1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP3 receptor mediated Ca2+ transients and Ca2+ insensitive phosphorylation of ERK1/2.     

Characterization of kinin receptors in human cultured detrusor smooth muscle cells

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.     

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.     

The use of kinin B1 and B2 receptor knockout mice and selective antagonists to characterize the nociceptive responses caused by kinins at the spinal level

The mechanisms by which kinins induce hyperalgesia in the spinal cord were investigated by using B(1) or B(2) knockout mice in conjunction with kinin selective agonists and antagonists. The i.t. administration of the kinin B(2) receptor agonists, bradykinin (BK) or Tyr(8)-BK produced dose-related thermal hyperalgesia evaluated in the hot-plate test. BK-induced hyperalgesia was abolished by the B(2) receptor antagonist Hoe 140. The i.t. injection of the kinin B(1) receptor agonists, des-Arg(9)-bradykinin (DABK) or des-Arg(10)-kallidin (DAKD) also caused dose-related thermal hyperalgesia. Different from the B(2) agonists, the i.t. injection of DABK or DAKD caused a weak, but prolonged hyperalgesia, an effect that was blocked by the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-bradykinin (DALBK). The i.t. injection of BK caused thermal hyperalgesia in wild-type mice (WT) and in the B(1) receptor knockout mice (B(1)R KO), but not in the B(2) receptor knockout mice (B(2)R KO). Similarly, the i.t. injection of DABK elicited thermal hyperalgesia in WT mice, but not in B(1)R KO mice. However, DABK-induced hyperalgesia was more pronounced in the B(2)R KO mice when compared with the WT mice. The i.t. injection of Hoe 140 or DALBK inhibited the second phase of formalin (F)-induced nociception. Furthermore, i.t. Hoe 140, but not DALBK, also inhibits the first phase of F response. Finally, the i.t. injection of DALBK, but not of Hoe 140, inhibits the long-term thermal hyperalgesia observed in the ipsilateral and in contralateral paws after intraplantar injection with complete Freund's adjuvant. These findings provide evidence that kinins acting at both B(1) and B(2) receptors at the spinal level exert a critical role in controlling the nociceptive processing mechanisms. Therefore, selective kinin antagonists against both receptors are of potential interest drugs to treat some pain states.     

Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes

UTP causes interleukin (IL)-6 production via mRNA expression through P2Y(2)/P2Y(4) receptors in human HaCaT keratinocytes. In the present study, we analyzed the mechanism of UTP-induced IL-6 production in these cells. UTP, an agonist of P2Y(2)/P2Y(4) receptors, induced phosphorylation of extracellular signal-regulated kinase (ERK) in a concentration- and time-dependent manner. PD98059, a MEK (mitogen-activated protein kinase kinase) inhibitor, and BAPTA-AM [O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester], an intracellular Ca(2+) chelator, reduced UTP-induced ERK phosphorylation and IL-6 mRNA expression. 2-APB [(2-aminoethoxy)diphenylborane], an inositol 1,4,5-trisphosphate (IP(3))-receptor antagonist, inhibited UTP-induced IL-6 mRNA expression; and the action of A23187, a Ca(2+) ionophore, resembled the action of UTP. In contrast, protein kinase C (PKC) downregulation and pertussis toxin did not affect UTP-induced IL-6 mRNA expression, suggesting that PKC and G(i) are not involved in the UTP-induced IL-6 production. However, AG1478, an epidermal growth factor (EGF)-receptor inhibitor, partially decreased UTP-induced ERK phosphorylation and IL-6 expression. These results suggest that UTP-induced IL-6 production is in part mediated via phosphorylation of ERK through G(q/11)/IP(3)/[Ca(2+)](i) and transactivation of the EGF receptor.     

The role of the epithelium in modulating the responses of guinea-pig trachea induced by bradykinin in vitro.

1. The effect of removing the epithelium on the responses of the guinea-pig isolated trachea (GPT) to bradykinin (BK) and prostaglandin E2 (PGE2) was investigated. 2. BK (3 pmol-10 nmol) induced dose-related relaxations of the intact (with epithelium), and contracted the rubbed (without epithelium) preparation of GPT. Similar responses were also obtained with PGE2 (0.3-3.0 nmol). 3. Indomethacin (1.4 microM) modified the BK-induced response of intact GPT, from a relaxation to a contraction, but inhibited the BK-induced contraction of the rubbed GPT. 4. There was a significant increase in PGE2 release from the intact GPT following stimulation with BK. 5. Removal of the epithelium from the GPT significantly reduced both basal and BK-induced generation of PGE2. 6. The induction of tone in the rubbed GPT by addition of acetylcholine (ACh) caused BK and PGE2 (0.3 nmol-3 nmol) to produce relaxations of the tissue. 7. Salbutamol (10(-8) M-10(-6) M) reduced the relaxations induced by BK on intact GPT, in a concentration-dependent manner. 8. These results suggest that both tone and an epithelial-dependent cyclo-oxygenase mechanism are important in modulating BK-induced responses of GPT.     

Differential regulation of bradykinin receptor density, intracellular Ca2+, and prostanoid release in skin and foreskin fibroblasts. Effects of cell density and interleukin-1alpha.

1. Bradykinin (BK) receptors, cytosolic Ca2+, and prostanoids were studied in human skin and foreskin fibroblasts. 2. Bmax values of BK receptors were higher in foreskin than in skin fibroblasts, increasing with cell densities in both cell types. IL-1alpha-dependent receptor induction was blocked by cycloheximide. 3. BK-stimulated cytosolic Ca2+ elevation was higher in confluent than in non-confluent cultures and larger in foreskin than in skin fibroblasts. Responses were not enhanced after IL-1-alpha-induced up-regulation of BK receptors. 4. Intrinsic prostanoid production was higher in foreskin than in skin fibroblasts at comparable cell densities. In foreskin, but not in skin fibroblasts, BK stimulation increased the release of PGE2 10 fold and that of 6-oxo-PGF1alpha 6-7 fold. 5. Preincubation with IL-1alpha had a marked effect on prostanoid release in foreskin fibroblasts only. Subsequent BK stimulation increased the release of PGE2 and 6-oxo-PGF1alpha 7-10 fold in skin fibroblasts while this increase was only 30% in foreskin fibroblasts. Release of TXA2 reached values up to one third of the other prostanoids. The IL-1alpha induced rise in BK-stimulated PGE2 synthesis was fully abolished by specific inhibition of cyclo-oxygenase 2. 6. IL-1alpha sensitized BK-stimulated prostanoid synthesis and modulated prostanoid patterns differently in fibroblasts from skin and foreskin. The IL-1alpha effects on prostanoid release were not related to BK receptor numbers nor to the BK-stimulated Ca2+ signal but appear to be due to induction of prostanoid synthesizing enzymes. Foreskin fibroblasts seem to be unique and significantly different from fibroblasts of other skin locations in respect to their response to inflammation-associated kinins and cytokines.     

Bradykinin-induced vascular responses in dog isolated lingual artery.

1. Kinin-induced vascular responses were studied and kinin receptor subtypes were characterized in canine isolated and preconstricted lingual arteries. 2. A low dose of bradykinin (BK; < 3 x 10(-14) mol) induced only vasodilation, while a higher dose of BK (> 3 x 10(-13) mol) frequently induced a biphasic response: a transient constriction followed by dilation. 3. The BK-induced vasodilation was mostly endothelium dependent but was also partly endothelium independent because although the dilation response was greatly reduced after removal of the endothelium, it was not completely abolished. 4. The dilation response to BK was significantly inhibited by the B2 kinin receptor antagonist HOE 140 and was partly reduced by indomethacin (10 mumol/L) (P < 0.05). 5. Bradykinin-induced vasoconstriction was enhanced in endothelium-denuded preparations. The constriction was significantly inhibited by HOE 140 (10(-10) mol/L). The BK-induced responses were not affected by the B1 kinin receptor antagonist des-Arg9-[Leu8]-BK (3 x 10(-11) mol/L). 6. The B1 kinin receptor agonist des-Arg9-BK (> 10(-12) mol/L) produced vasodilation in 60% of endothelium-intact preparations. In 20% of the endothelium-intact preparations des-Arg9-BK produced a biphasic response: weak vasoconstriction followed by weak vasodilation. The des-Arg9-BK-induced dilation and constriction were significantly inhibited by des-Arg9-[Leu8]-BK (3 x 10(-11) mol/L), but were not affected by HOE 140 (10(-10) mol/L). 7. In conclusion, it appears that both B1 and B2 kinin receptors are present in the dog lingual artery. Both receptor subtypes mediate either vasodilation or vasoconstriction and BK-induced vasodilation is mostly endothelium dependent, although it may also be partially prostaglandin dependent.     

Heterologous desensitization of bradykinin-induced phosphatidylinositol response and Ca2+ mobilization by neurotensin in NG108-15 cells

The heterologous desensitization of the bradykinin (BK)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) by neurotensin was studied in neuroblastoma x glioma hybrid NG108-15 cells. The addition of neurotensin to the cells resulted in an increase in [Ca2+]i and an increase in the formation of inositol phosphates in Ca2+-free medium. Pretreatment of the cells with neurotensin resulted in 43% decrease in the BK-induced increase of [Ca2+]i. The increase in [Ca2+]i induced by ionomycin, which causes Ca2+ release from the intracellular pool, was not decreased by pretreatment with neurotensin. This indicates that the inhibitory effect of neurotensin on the BK-induced increase of [Ca2+]i was not due to depletion of the intracellular Ca2+ pool. Pretreatment with neurotensin also caused a 47% decrease in the BK-induced formation of inositol trisphosphates (IP3). This decrease was not due to depletion of phosphatidylinositol bisphosphates. Neurotensin did not inhibit [3H]BK binding to cell membranes. These results show that neurotensin desensitizes the BK responses of NG108-15 cells, heterologously, perhaps by changes in phospholipase C and/or guanine nucleotide-binding protein (G-protein).