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 Ca2+ waves that rely on Ca2+ 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 Ca2+ waves in ICC. Intracellular [Ca2+] 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 mu M) significantly inhibited the generation of spontaneous Ca2+ waves in ICC and this was associated with a significant decrease in the ER Ca2+ load, measured with 10 mM caffeine responses. Ca2+ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 10 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP(3)Rs) with 10 mu M phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca2+ 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 Ca2+ load sufficient to inactivate IP(3)Rs but not RyRs. (C) 2014 Elsevier Ltd. All rights reserved.