The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca²⁺ waves in interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca(2+) waves that rely on Ca(2+) release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca(2+) waves in ICC. Intracellular [Ca(2+)] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca(2+) waves in ICC and this was associated with a significant decrease in the ER Ca(2+) load, measured with 10mM caffeine responses. Ca(2+) waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP3Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca(2+) wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca(2+) load sufficient to inactivate IP3Rs but not RyRs.