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Opposing functional effects of cyclic GMP and cyclic AMP may act through protein phosphorylation in rabbit cardiac
1Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635, USA.
Abstract:
1. We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) oppose the positive effects of cyclic AMP (cAMP) in cardiac myocytes through interaction at the level of their respective protein kinases. 2. Cell shortening was studied using a video-edge detector. The O2 consumption of a suspension of rabbit ventricular myocytes was measured using O2 electrodes. Protein phosphorylation was measured autoradiographically following 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-cAMP 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 microm s(-1). 8-Br-cAMP 10(-5) M increased Pcs (from 3.7+/-0.2 to 4.8+/-0.2) and 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. Following 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 5720 decreased the elevated Pcs and Rs significantly and KT 5823 10(-6) M further 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-cAMP 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 their respective protein kinases.
Insights
Cyclic GMP (cGMP) and cyclic AMP (cAMP) signaling pathways interact at the protein kinase level in cardiac myocytes. This interaction explains their opposing effects on heart cell function.
Area of Science:
- Cardiology
- Molecular Biology
- Cell Physiology
Background:
- Cyclic nucleotides like cyclic AMP (cAMP) and cyclic GMP (cGMP) play critical roles in regulating cardiac function.
- Understanding the interplay between cAMP and cGMP signaling is essential for elucidating cardiac myocyte behavior.
Purpose of the Study:
- To investigate the hypothesis that cGMP antagonizes cAMP's effects in cardiac myocytes.
- To determine if this opposition occurs at the level of their respective protein kinases.
Main Methods:
- Utilized rabbit ventricular myocytes to study cell shortening via video-edge detection.
- Measured O2 consumption using O2 electrodes.
- Assessed protein phosphorylation patterns using SDS-PAGE and autoradiography.
- Administered specific analogs of cGMP and cAMP, along with protein kinase inhibitors (KT 5720 for cAMP-dependent protein kinase and KT 5823 for cGMP-dependent protein kinase).
Main Results:
- cGMP analogs decreased cell shortening and contraction rate, while cAMP analogs increased these parameters.
- Pre-treatment with cGMP analogs diminished the positive effects of cAMP analogs on cell shortening.
- Inhibition of cAMP-dependent protein kinase potentiated cGMP's negative effects, whereas inhibition of cGMP-dependent protein kinase reversed cGMP's effects.
- Both cGMP and cAMP induced phosphorylation of the same five protein bands, suggesting shared or interacting targets.
Conclusions:
- The opposing functional effects of cGMP and cAMP in cardiac myocytes are mediated through interactions at the level of their respective protein kinases.
- This study provides molecular insights into the antagonistic roles of these cyclic nucleotides in cardiac physiology.