Ca2+‐dependent and ‐independent mechanism of cyclic‐AMP reduction: mediation by bradykinin B2 receptors

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Abstract
1 Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 ±4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 ±1.1 nM). 2 The Ca2+ ionophore, ionomycin, mimicked both the bradykinin-induced transient reduction in the forskolin-stimulated cyclic AMP level and the sustained reduction in the isoprenaline-stimulated cyclic AMP level. 3 The Ca2+-dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin. 4 The involvement of calmodulin-dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin-induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level. 5 Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca2+-independent activation of phosphodiesterase activity by bradykinin in DDT1 MF-2 cells. 6 The bradykinin B1 receptor agonist, desArg9–bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK) and D-Arg[Hyp3, Thi5–8, D-Phe7]-BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells. 7 Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK. The bradykinin B2 receptor agonist, desArg9–bradykinin, did not evoke a rise in cytoplasmic Ca2+. 8 It is concluded, that stimulation of bradykinin B2 receptors causes a reduction in cellular cyclic AMP in DDT1 MF-2 cells. This decline in cyclic AMP is partly mediated by a Ca2+/calmodulin independent activation of phosphodiesterase activity. The increase in [Ca2+]i mediated by bradykinin B2 receptors inhibited forskolin- and isoprenaline-activated adenylyl cyclase differently, most likely by interfering with different components of the adenylyl cyclase signalling pathway.