Opposing functional effects of cyclic GMP and cyclic AMP may act through protein phosphorylation in rabbit cardiac myocytes

1 We tested the hypothesis that the negative functional effects of cyclic G MP (cGMP) oppose the positive effects of cyclic AMP (cAMP) in cardiac myocy tes through interaction at the level of their respective protein kinases. 2 Cell shortening was studied using a video-edge detector. The O-2 consumpt ion of a suspension of rabbit ventricular myocytes was measured using O-2 e lectrodes. Protein phosphorylation was measured autoradiographically follow ing SDS-PAGE. Data were collected with: (1) 8-bromo-cGMP (8-Br-cGMP) 10(-7) or 10(-5) m; (2) 8-bromo-cAMP (8-Br-cAMP) 10(-7) or 10(-5) m; (3) 8-Br-cAM P 10(-5) m followed by 8-Br-cGMP 10(-7) or 10(-5) m; (4) 8-Br-cGMP 10(-5) m followed by 8-Br-cAMP 10(-7) or 10(-5) m; (5) 8-Br- cGMP 10(-7) or 10(-5) m followed by KT 5720 (cAMP- dependent protein kinase inhibitor) or KT 5823 (cGMP-dependent protein kinase inhibitor) 10(-6) m; and (6) 8-Br-cAMP 10(- 7) or 10(-5) m followed by KT 5720 or KT 5823 10-6 M. 3 8-Br-cGMP 10(-5) M decreased percent shortening (Pcs) from 6.3 +/-0.6 to 3.6 +/-0.4% and rate of shortening (Rs) from 66.7 +/-4.4 to 41.8 +/-4.2 mum s(-1). 8-Br-cAMP 10(-5) m increased Pcs (from 3.7 +/-0.2 to 4.8 +/-0.2) an d Rs (from 50.0 +/-3.0 to 60.0 +/-3.1). With 8-Br-cAMP 10(-5) m, 8-Br-cGMP 10(-5) m decreased Pcs and Rs less. The positive functional effects of 8-Br -cAMP 10(-7) or 10(-5) m were also diminished with 8-Br-cGMP 10(-5) m. Foll owing 8-Br-cGMP 10(-7) or 10(-5) m, KT 5720 10(-6) m further decreased Pcs to 2.5 +/-0.3 and Rs to 30.0 +/-4.1. KT 5823 10(-6) m returned Pcs to 4.7 /-0.4 and Rs to 61.3 +/-5.3. Following 8-Br-cAMP 10(-7) or 10(-5) m, KT 572 0 decreased the elevated Pcs and Rs significantly and KT 5823 10(-6) m furt her increased these parameters. 4 cGMP and cAMP phosphorylated the same five protein bands. With KT 5720 or KT 5823, all of the bands were lighter at the same concentration of 8-Br-c AMP and 8-Br-cGMP. 5 We conclude that, in rabbit ventricular myocytes, the opposing functional effects of cGMP and cAMP are related to the interaction at the level of th eir respective protein kinases.