The relative contributions of Ca(2+)-induced Ca(2+) release (CICR) versus Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal Ca(2+) images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in several small, discrete areas just beneath the cell membrane. These Ca(2+) "hot spots" then spread slowly through the myoplasm as Ca(2+) waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca(2+) sparks, which declined quickly. The number of Ca(2+) sparks, or hot spots, was closely related to the depolarization duration in the range of approximately 5-20 ms. There was an apparent threshold depolarization duration of approximately 10 ms within which to induce enough Ca(2+) transients to spread globally and then induce a contraction. Application of 100 microM ryanodine to the pipette solution did not change the resting [Ca(2+)](i) or the VDCC current, but it did abolish Ca(2+) hot spots elicited by depolarization. Application of 3 microM xestospongin C reduced ACh-induced Ca(2+) release but did not affect depolarization-induced Ca(2+) events. The addition of 100 microM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca(2+)](i) rise triggered by a single action potential is not due mainly to Ca(2+) influx through VDCCs but is attributable to the subsequent two-step CICR.

The possibility that the ryanodine receptor type 2 (RyR2) can function as the major Ca 2+ -induced Ca 2+ release (CICR) channel in excitation-contraction (E-C) coupling was examined in smooth muscle cells (SMCs) isolated from urinary bladder (UB) of RyR2 heterozygous KO mice (RyR2 +/− ). RyR2 mRNA expression in UB from RyR2 +/− was much lower than that in wild-type (RyR2 +/+ ). In single UBSMCs from RyR2 +/+ , membrane depolarization under voltage clamp initially induced several local Ca 2+ transients (hot spots) in peripheral areas of the cell. Then, Ca 2+waves spread from Ca 2+ hot spots to other areas of the myocyte. The number of Ca 2+ hot spots elicited by a short depolarization (< 20 ms) in UBSMCs of RyR2 +/− was significantly smaller than in those of RyR2 +/+ . The force development induced either by direct electrical stimulation or by 10 μM acetylcholine in tissue segments of RyR2 +/− was smaller than and comparable to those in RyR2 +/+ , respectively. The frequency of spontaneous transient outward currents in single myocytes and the membrane depolarization by 1 μM paxilline in tissue segments from RyR2 +/− were significantly lower and smaller than those in RyR2 +/+ , respectively. The urination frequency and volume per voiding in RyR2 +/− were significantly increased and reduced, respectively, compared with RyR2 +/+ . In conclusion, RyR2 plays a crucial role in the regulation of CICR during E-C coupling and also in the regulation of resting membrane potential, presumably via the modulation of Ca 2+ -dependent K + channel activity in UBSMCs and, thereby, has a pivotal role in the control of bladder activity.

Abstract. We examined effects of small and intermediate conductance Ca2+ -activated K + (SK and IK) channel openers, DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) and NS309 (3-oxime-6,7-dichloro-1H-indole-2,3-dione), on L-type Ca 2+ channel current (I Ca ) that was measured in smooth muscle cells isolated from mouse urinary bladder under whole cell voltage-clamp. The I Ca was concentration-dependently inhibited by DCEBIO and NS309; half inhibition was obtained at 71.6 and 10.6 µM, respectively. The specificity of NS309 to the IK channel over the Ca 2+ channel appears to be high and higher than that of DCEBIO. DCEBIO and even NS309 may, however, substantially block Ca 2+ channels when used as SK channel openers.

No abstract