Signalling pathways of bradykinin-mediated arachidonic acid release in MDCK-D1 cells.

An investigation was undertaken to elucidate the signal transduction pathways involved in bradykinin (BK)-mediated release of arachidonic acid (AA) from the D1 clone of Madin-Darby canine kidney cells (MDCK-D1) which display distal tubule- and cortical collecting duct principal cell-like characteristics. Prostaglandins (PG), generated subsequent to BK stimulation, are known to modulate arginine-vasopressin (AVP)-stimulated water flow across this portion of the nephron. The enzyme immediately responsible for AA release was determined to be the 85 kDa cytosolic phospholipase A$\sb2$ (cPLA$\sb2),$ since Western blots revealed the presence of this enzyme and its specific inhibition by an arachidonate analogue completely blunted BK-stimulated AA release. Additionally, in vitro PLA$\sb2$ activity could be significantly reduced by preincubating cell lysates with an antibody to this enzyme. Lastly, this in vitro activity met all the requirements specific to cPLA$\sb2,$ including activity at micromolar Ca$\sp{2+}$ concentrations and dithiotreitol (DTT)-insensitivity. The findings herein suggest the signalling route taken for BK-induced AA release involves phosphatidyicholine-specific phospholipase C (PC-PLC) as well as phospholipase D (PLD). Accordingly, production of sn-1,2-diacylglycerol (DAG) and to a lesser extent, phosphatidic acid (PA), both contribute to this release of AA by enhancing PLA$\sb2$ activity within the cellular membranes, whereas the activation of phosphatidylinositol-specific phopholipase C (PI-PLC) and subsequent inositol trisphosphate (InsP$\sb3$) production does not. While reports indicate the activation of protein kinase C (PKC) is required for epinephrine-mediated AA release in this cell line, inhibition of PKC failed to abrogate BK-stimulated AA release. On the other hand, down regulation of PKC levels via long-term incubation with phorbol ester (PMA) reduced both BK- and calcium ionophore (A23187)-induced AA release. However, both in vitro cPLA$\sb2$ activity and its phosphorylation, but not its expression, were significantly reduced subsequent to long-term PMA treatment, thereby demonstrating that this strategy falsely implicates immediate activation of PKC as being required for BK-mediated AA release. Extracellular calcium (Ca$\sp{2+})$ was also needed for AA release as blockade of receptor-operated Ca$\sp{2+}$ channels significantly decreased BK-induced AA release. In addition, a negative regulatory pathway in MDCK cells was demonstrated which diminishes BK-mediated AA release. Agents which cause elevations in adenosine-3$\sp\prime,5\sp\prime$-cyclic monophosphate (cAMP) levels, such as AVP or forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX), were found capable of significantly reducing BK-induced AA release and in vitro PLA$\sb2$ activity. This method of inhibition could represent a physiological mechanism of negative feedback promoted by agents which increase cAMP levels within the cells of the distal tubule and collecting duct. Possible targets for inhibition are suggested in light of results obtained in the present thesis and reported by others