Regulation of KCa-channel activity by cyclic ADP-ribose and ADP-ribose in coronary arterial smooth muscle.

Abstract

The enzymatic pathway responsible for the production and metabolism of cyclic ADP-ribose (cADP-R) in small bovine coronary arteries was characterized, and the role of cADP-R and ADP-ribose (ADP-R) in the regulation of the activity of large-conductance Ca2+-activated K+ (KCa) channels was determined in vascular smooth muscle cells (SMC) prepared from these vessels. We found that cADP-R and ADP-R were produced when the coronary arterial homogenates were incubated with 1 mM beta-NAD. The time course of the enzyme reactions showed that the maximal conversion rate (1.37 +/- 0.03 nmol . min-1 . mg protein-1) of beta-NAD to cADP-R was reached after 3 min of incubation. As incubation time was prolonged, the production of ADP-R was increased to a maximal rate of 3.66 +/- 0.03 nmol . min-1 . mg protein-1, whereas cADP-R production decreased. Incubation of the homogenate with cADP-R produced a time-dependent increase in the synthesis of ADP-R. Comparison of coronary arterial microsomes with cytosols shows that the production of both cADP-R and ADP-R in microsomes was significantly greater. In excised inside-out membrane patches of single coronary SMC, the KCa channels were activated when beta-NAD, the precursor for both cADP-R and ADP-R, was applied to the internal surface. This effect of beta-NAD may be associated with the production of ADP-R, because the KCa-channel activity was increased by ADP-R in a concentration-dependent manner. The open-state probability of the KCa channels increased from a control level of 0.08 +/- 0.03 to 0.17 +/- 0.05 even at the lowest ADP-R concentration (0.1 microM) studied. However, cADP-R reduced the KCa-channel activity, and the threshold concentration of cADP-R that decreased the average channel activity of the KCa channels was 1 microM. These results provide evidence that cADP-R is produced and metabolized in the coronary arterial smooth muscle and that a cADP-R/ADP-R pathway participates in the control of the KCa-channel activity in vascular SMC.