ABSTRACT
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.
Subject(s)
Bradykinin/analogs & derivatives , Calcium/metabolism , Endothelial Cells/drug effects , Signal Transduction , Vasodilator Agents/pharmacology , Animals , Bradykinin/pharmacology , Calcium-Transporting ATPases/antagonists & inhibitors , Calcium-Transporting ATPases/metabolism , Cell Line, Transformed , Diglycerides/pharmacology , Endothelial Cells/enzymology , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Male , Mice , Pyrrolidinones/pharmacology , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Bradykinin B2/drug effects , Receptor, Bradykinin B2/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases , TRPC Cation Channels/drug effects , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , TRPC6 Cation Channel , Thapsigargin/pharmacology , Time Factors , Transfection , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolismABSTRACT
TRPC channels are a group of Ca(2+)-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca(2+) influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3-6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure.
Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Ion Channels/biosynthesis , Muscle, Smooth, Vascular/metabolism , Adolescent , Adult , Aged , Calcium Channels/biosynthesis , Cation Transport Proteins/biosynthesis , Cerebral Arteries/metabolism , Coronary Vessels/metabolism , Endothelium, Vascular/cytology , Female , Gene Expression , Humans , Immunohistochemistry , In Situ Hybridization , Male , Membrane Proteins/biosynthesis , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , TRPC Cation Channels , TRPC6 Cation Channel , TRPM Cation ChannelsABSTRACT
Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+ influx of rat aortic endothelial cells in response to flow. We found that the Ca2+ signaling in response to flow could be greatly influenced by the status of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin (4 micromol/L) or cyclopiazonic acid (10 micromol/L) drastically sensitized the Ca2+ influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 micromol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+ stores. In addition, we found that depletion-sensitized Ca2+ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+ status in determining the Ca2+ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+ influx.
