DUAL BRADYKININ B-2 RECEPTOR SIGNALING IN A431 HUMAN EPIDERMOID CARCINOMA-CELLS - ACTIVATION OF PROTEIN-KINASE-C IS COUNTERACTED BY A G(S)-MEDIATED STIMULATION OF THE CYCLIC-AMP PATHWAY

Cell membranes of the human epidermoid cell line A431 express classica l bradykinin (BK) B-2 receptors, as assessed by [H-3]BK binding studie s. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation ( t(1/2) approximate to 1 min) is followed by a slow inhibition (t(1/2) approximate to 20 min) of PKC translocation measured by [H-3]phorbol 1 2,13-dibutyrate binding. In addition, BK stimulated both adenylate cyc lase activity in A431 membranes and accumulation of intracellular cycl ic AMP (cAMP) in intact cells in a retarded manner. A possible BK-indu ced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was f ound in A431 membranes to be specifically phosphorylated in the presen ce of both BK and protein kinase A (PKA). An anti-alpha(s)-antibody, A S 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involv ement of G(s) proteins in the BK action. The BK-activated cAMP signall ing system might be important for the observed inactivation of PKC slo wly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an i nhibitor of PKA, adenosine 3'. 5'-monophosphothioate (cyclic, Rp isome r). The inhibition of PKC translocation might be exerted directly at t he level of PKC activation, since stimulation of phosphoinositide hydr olysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thu s our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single cl ass of B-2 receptors but two different G proteins. The lagging stimula tion of the cAMP signalling pathway via G(s) might serve to switch off PKC, which is rapidly activated via G(q)-mediated stimulation of phos phoinositide hydrolysis.