REGULATION OF BRADYKININ-STIMULATED PHOSPHOLIPASE-C AND ARACHIDONIC-ACID RELEASE BY PROTEIN-KINASE-A IN MDCK-D1 CELLS

Regulation of phospholipases C (PLC) and arachidonic acid (AA) release by cAMP-dependent protein kinase (PKA) was investigated in MDCK-D1 ce lls. Bradykinin (BDK) was used to stimulate PLC and AA release, while arginine vasopressin (AVP), forskolin (FSK), isobutylmethylxanthine (I BMX) were used to increase cAMP levels and stimulate PKA. When cells w ere preincubated for 20 min with 10 mu M FSK + 0.5 mM IBMX, and subseq uently treated with 1 mu M BDK or control medium for 10 min, the basal and BDK-stimulated PLC activity, measured as accumulated labelled ino sitol phosphate (InsP) after 40 min and inositol trisphosphate (InsP(3 )) after 10 s, were significantly inhibited. In a parallel manner, FSK + IBMX also significantly decreased both basal and BDK-stimulated dia cylglycerol (DAG) production. The basal and BDK-enhanced AA release in to the media was also significantly inhibited by pretreatment with FSK + IBMX. In parallel experiments, H-89, a specific inhibitor of PKA, w as preincubated for 60 min prior to addition of BDK and this resulted in a reversal of FSK + IBMX-induced inhibition of basal and BDK-stimul ated PLC activity and AA release. An inhibitor of inositide-hydrolysin g PLC, U73122, (1 mu M) was also found to blunt BDK-stimulated PLC act ivity and BDK-enhanced AA release which indicated that stimulation of AA release by the nonapeptide was second to PLC activation. The ionoph ore, A23187, (10 mu M) greatly stimulated AA release and to a much les ser extent, PLC activity. Its effect on AA release however was not blo cked by inhibiting protein kinase C (PKC) with staurosporine (SSP) and consequently did not notably involve the PLC-PKC cascade. Activation of PKA with FSK + IBMX was found to significantly inhibit the enhancem ent of AA release by ionophore. With 12-tetradecanoyl-phorbol-13-aceta te (TPA) also present there was a synergistic increase in the A23187-s timulated AA release and activation of PKA under such conditions inhib ited AA release to a similar extent though the synergistic effect rema ined. The results strongly suggest a role for PKA in the regulation of PLC activity and AA release in MDCK-D1 cells. Control of AA release b y PKA, is mediated both by mechanisms which involve blunting of PLC ac tivity and mechanisms which are downstream from the PLC-PKC cascade.