thapsigargin and icatibant

ATP has broad functions as an autocrine/paracrine molecule. The mode of ATP release and its intracellular source, however, are little understood. Here we show that bradykinin via B(2)-receptor stimulation induces the extracellular release of ATP via the inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]-signaling pathway in cultured taenia coli smooth muscle cells. It was found that bradykinin also increased the production of Ins(1,4,5)P(3) and 2-APB-inhibitable [Ca(2+)](i). The evoked release of ATP was suppressed by the Ca(2+)-channel blockers, nifedipine, and verapamil. Moreover, the extracellular release of ATP was elicited by photoliberation of Ins(1,4,5)P(3). Bradykinin caused a quick and transient accumulation of intracellular ATP from cells treated with 1% perchloric acid solution (PCA), but not with the cell lysis buffer. Peak accumulation was prevented by 2-APB and thapsigargin, but not by nifedipine or verapamil, inhibitors of extracellular release of ATP. These findings suggest that bradykinin elicits the extracellular release of ATP that is mediated by the Ins(1,4,5)P(3)-induced Ca(2+) signaling and, finally, leads to a Ca(2+)-dependent export of ATP from the cells. Furthermore, the bradykinin-induced transient accumulation of ATP in the cells treated with PCA may imply a possible release of ATP from the endoplasmic reticulum.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Benzyl Compounds; Boron Compounds; Bradykinin; Calcium; Calcium Channel Blockers; Cells, Cultured; Colon; Egtazic Acid; Endoplasmic Reticulum; Estrenes; Ethylmaleimide; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Male; Microscopy, Confocal; Muscle, Smooth; Nifedipine; Pyrrolidinones; Receptor, Bradykinin B2; Rotenone; Thapsigargin; Thiazolidines; Verapamil

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca(2+) (Ca(2+))(i) in endothelial cells, resulting in Ca(2+)-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca(2+) influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca(2+) influx was resolved into capacitative Ca(2+) entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 microM) and a phospholipase C (PLC) inhibitor (U73122; 10 microM). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.

Topics: Animals; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Line, Transformed; Diglycerides; Endothelial Cells; Enzyme Inhibitors; Estrenes; Male; Mice; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; RNA Interference; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin; Time Factors; Transfection; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases; Vasodilator Agents

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.

Topics: Animals; Bradykinin; Bradykinin Receptor Antagonists; Calcium; Calcium Channel Blockers; Cells, Cultured; Cholera Toxin; Dogs; Enzyme Inhibitors; Epithelial Cells; Estrenes; Female; Hydrolysis; Imidazoles; Male; Pertussis Toxin; Phosphatidylinositols; Pyrrolidinones; Receptor, Bradykinin B2; Thapsigargin; Trachea; Type C Phospholipases; Virulence Factors, Bordetella

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.

Topics: Angiotensin II; Animals; Bradykinin; Calcium; Cell Line; Enzyme Activation; Kallidin; Liver; Rats; Receptor, Bradykinin B2; Receptors, Bradykinin; Thapsigargin

1. The contribution of sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA)-regulated Ca2+ stores to the increase in intracellular free calcium ([Ca2+]i) induced by bradykinin (BK) was investigated in fura-2 loaded human tracheal smooth muscle cells (TSMC). For this purpose, we used thapsigargin, a selective inhibitor of Ca(2+)-ATPases of intracellular organelles. 2. Thapsigargin (10(-9) to 10(-6) M) induced a dose-dependent increase in [Ca2+]i in the presence of external Ca2+ with an EC50 value of 7.33 +/- 1.26 nM. In Ca(2+)-free conditions, the addition of Ca2+ (1.25 mM) caused an increase in [Ca2+]i which was directly proportional to the pre-incubation time of the cells with thapsigargin. Net increases of 60 +/- 9, 150 +/- 22 and 210 +/- 27 nM were obtained after 1, 3 and 5 min, respectively. 3. In the presence of extracellular Ca2+, BK induced a typical biphasic increase in [Ca2+]i with a fast transient phase and a sustained phase. The sustained component was reversed by addition of a bradykinin B2-receptor antagonist (Hoe 140, 10(-6) M) to the buffer as well as by deprivation of Ca2+. The transient phase induced by BK, histamine and carbachol was inhibited in a time-dependent way by preincubation of the cells with thapsigargin. 4. Comparative western blotting of human TSMC membranes using anti-SERCA2 isoform-specific antibodies clearly showed the greater expression of the 100-kDa SERCA2-b isoform compared with the SERCA2-a isoform. 5. Our data show that thapsigargin-sensitive Ca2+ stores contribute significantly to the activation of human TSMC which suggests a role for these stores in the subsequent induction of Ca2+ influx. These stores appear to be controlled by the Ca2+-ATPases (SERCA2-b isoform) which could also participate in the regulation of Ca2+ influx through the plasma membrane.

Topics: Biological Transport; Bradykinin; Bradykinin Receptor Antagonists; Calcium; Calcium-Transporting ATPases; Carbachol; Cells, Cultured; Enzyme Inhibitors; Histamine; Humans; Muscle, Smooth; Receptor, Bradykinin B2; Sarcoplasmic Reticulum; Terpenes; Thapsigargin; Trachea