Intracellular Ca2+ ([Ca2+]i) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca2+ release from intracellular Ca2+ stores produces [Ca2+]i oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca2+]i oscillations in ICCs are preserved in the presence of dihydropyridine Ca2+ antagonists, which suppress Ca2+ activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca2+]i oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca2+]i oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP3Rs. The reconstituted [Ca2+]i oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

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 the effects of methyl-β-cyclodextrin (MβCD) on Ca

The Na(+)/Ca(2+) exchanger (NCX) is thought to be a key molecule in the regulation of cytosolic Ca(2+) dynamics. The relative importance of the two Ca(2+) transport modes of NCX activity leading to Ca(2+) efflux (forward) and influx (reverse) in smooth muscle, however, remains unclear. Unexpectedly, spontaneous contractions of urinary bladder smooth muscle (UBSM) were enhanced in transgenic mice overexpressing NCX1.3 (NCX1.3(tg/tg)). The enhanced activity was attenuated by KB-R7943 or SN-6. Whole cell outward NCX current sensitive to KB-R7943 or Ni(2+) was readily detected in UBSM cells from NCX1.3(tg/tg) but not wild-type mice. Spontaneous Ca(2+) transients in myocytes of NCX1.3(tg/tg) were larger and frequently resulted in propagating events and global elevations in cytosolic Ca(2+) concentration. Significantly, NCX1.3(tg/tg) mice exhibited a pattern of more frequent urination of smaller volumes and this phenotype was reversed by oral administration of KB-R7943. On the other hand, KB-R7943 did not improve it in KB-R7943-insensitive (G833C-)NCX1.3(tg/tg) mice. We conclude that NCX1.3 overexpression is associated with abnormal urination owing to enhanced Ca(2+) influx via reverse mode NCX leading to prolonged, propagating spontaneous Ca(2+) release events and a potentiation of spontaneous UBSM contraction. These findings suggest the possibility that NCX is a candidate molecular target for overactive bladder therapy.

The activated B-cell diffuse large B-cell-like lymphoma (ABC-DLBCL) correlates with poor prognosis. The B-cell receptor signaling pathway is known to be dysregulated in NHL/CLL and given BTK is a downstream mediator of BCR signaling, BTK constitutes an interesting and obvious therapeutic target. Given the high potency and selectivity of the BTK inhibitor, ONO/GS-4059, it was hypothesized that, the anti-tumor activity of ONO/GS-4059 could be further enhanced by combining it with the anti-CD20 Abs, rituximab (RTX) or obinutuzumab (GA101). ONO/GS-4059 combined with GA101 or RTX was significantly better than the respective monotherapy with tumor growth inhibition (TGI) of 90% for the GA101 combination and 86% for the RTX combination. In contrast, ibrutinib (PCI-32765) combined with RTX did not result in improved efficacy compared with respective monotherapy. Taken together these data indicate that the combination of ONO/GS-4059 with rituximab and particularly obinutuzumab may be an effective treatment for ABC-DLBCL.