CYCLOPIAZONIC ACID, INHIBITING THE ENDOPLASMIC-RETICULUM CALCIUM-PUMP, REDUCES THE CANINE COLONIC PACEMAKER FREQUENCY

The slow wave frequency of the canine colon has previously been hypoth esized to be paced by an intracellular biochemical clock. We investiga ted the relationship between the endoplasmic reticulum (ER) Ca++ and t he periodicity of the biochemical clock. Cyclopiazonic acid, a specifi c inhibitor of the ER Ca++ pump, dose-dependently decreased the pacema ker frequency. Similarly, chelating cytosolic Ca++ with bis-(o-aminoph enoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) also decreased the p acemaker frequency. These observations suggest that delaying the Ca+uptake into the ER decreases the pacemaker frequency. The pacemaker fr equency was similarly decreased by neomycin [inhibiting inositol 1,4,5 -triphosphate (IP3) synthesis] and by caffeine at concentrations highe r than 5 mM (inhibiting the IP3-sensitive Ca++ channels in the ER memb rane). Hence the IP3-sensitive Ca++ stores are involved in the biochem ical clock. Ryanodine (up to 60 mu M) did not affect the pacemaker fre quency, which indicates that a ryanodine-sensitive store, if it exists , is not coupled to the biochemical clock. Electron microscopy showed that the smooth ER forms an extensive network of subsurface cisternae that is closely associated with large areas of the cytoplasmic face of the plasma membrane. These structures were the most extensive in inte rstitial cells of Cajal, slightly less in branching smooth muscle cell s and far less in circular muscle cells. In summary, on the basis of t hese electrophysiological and morphological observations, we hypothesi ze that the Ca++ refilling cycle of the IP3-sensitive calcium stores a ssociated with the plasma membrane determines the frequency of the pac emaker activity generated by the submuscular interstitial cells of Caj al-smooth-muscle network of the canine colon.