Activation of cAMP-Dependent Protein Kinase in Rat Aorta by cAMP Analogs Is Not Correlated with Relaxation

Abstract

The role of cAMP-dependent protein kinase (PKA) in the relaxation of vascular smooth muscle by cAMP analogs was studied. The analog N⁶,2’-O-dibutyryl-cAMP (dbu-cAMP) reduced KC1- and phenylephrine (PE)-induced tension in rat aortic rings in a dose-dependent fashion (10–100 µM). Conversely, incubation with 8-bromo-cAMP (8Br-cAMP; 10-100 µM)had very little effect on tension. The soluble and particulate PKA activity was determined in the analog-treated, PE-contracted tissue. Interference from extracellular analogs was eliminated by washout of analog prior to homogenization of the tissue for the PKA assay and by the addition of charcoal (10 mg/ml) to the homogenization buffer. The nonrelaxant analog 8Br-cAMP significantly increased the activity ratio of soluble PKA at a concentration of 30 µM and, in the particulate fraction, increased the activity ratio at 30- and 100 µM. Total PKA activity in the soluble fraction was significantly decreased by all concentrations of 8Br-cAMP used (10, 30, 100 µM). The relaxant analog dbu-cAMP had no significant effect at any concentration on catalytically active or total PKA activity levels. The only effect seen, at 30 µM, was an increase in the particulate activity ratio, and this was smaller than that seen with 8Br-cAMP. The basis for the alteration in PKA activity by 8Br-cAMP was a decrease in total soluble PKA activity as opposed to an increase in free catalytic PKA subunit. The fall in total activity was most likely due to adsorption by charcoal of catalytic subunit released from the 8Br-cAMP-activated holoenzyme. Thus, under the conditions used to assay PKA activity in cAMP-analog-treated vascular tissue, a decrease in total PKA activity represents an activation of the enzyme and, in fact, appears to be a better indicator of activation than changes in activity ratios. In either case, activation of soluble and particulate PKA by cAMP analogs did not correlate with relaxation of rat aortic rings.