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Biochimica et biophysica acta

Regulation of bradykinin-stimulated phospholipase C and arachidonic acid release by protein kinase A in MDCK-D1 cells.


PMID 7548185

Abstract

Regulation of phospholipases C (PLC) and arachidonic acid (AA) release by cAMP-dependent protein kinase (PKA) was investigated in MDCK-D1 cells. Bradykinin (BDK) was used to stimulate PLC and AA release, while arginine vasopressin (AVP), forskolin (FSK), isobutylmethylxanthine (IBMX) were used to increase cAMP levels and stimulate PKA. When cells were preincubated for 20 min with 10 microM FSK + 0.5 mM IBMX, and subsequently treated with 1 microM BDK or control medium for 40 min, the basal and BDK-stimulated PLC activity, measured as accumulated labelled inositol phosphate (InsP) after 40 min and inositol trisphosphate (InsP3) after 10 s, were significantly inhibited. In a parallel manner, FSK + IBMX also significantly decreased both basal and BDK-stimulated diacylglycerol (DAG) production. The basal and BDK-enhanced AA release into the media was also significantly inhibited by pretreatment with FSK + IBMX. In parallel experiments, H-89, a specific inhibitor of PKA, was preincubated for 60 min prior to addition of BDK and this resulted in a reversal of FSK+IBMX-induced inhibition of basal and BDK-stimulated PLC activity and AA release. An inhibitor of inositide-hydrolysing PLC, U73122, (1 microM) was also found to blunt BDK-stimulated PLC activity and BDK-enhanced AA release which indicated that stimulation of AA release by the nonapeptide was second to PLC activation. The ionophore, A23187, (10 microM) greatly stimulated AA release and to a much lesser extent, PLC activity. Its effect on AA release however was not blocked 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 enhancement of AA release by ionophore. With 12-tetradecanoyl-phorbol-13-acetate (TPA) also present there was a synergistic increase in the A23187-stimulated AA release and activation of PKA under such conditions inhibited AA release to a similar extent though the synergistic effect remained. 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 by PKA, is mediated both by mechanisms which involve blunting of PLC activity and mechanisms which are downstream from the PLC-PKC cascade.

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84121903
MDCK Cell Line canine