Two-step Ca²⁺ intracellular release underlies excitation-contraction coupling in mouse urinary bladder myocytes

Abstract

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.

Regulation of Ryanodine Receptor–Mediated Ca2+ Release in Vas Deferens Smooth Muscle Cells

“…Since the up-regulation of L-type VDCC (α1C) expression by testosterone has been reported in coronary arterial SMCs (31), the decrease in functional expression of Ca 2+ channels in Flu was expected. However, the observation that the [Ca The significant contribution of CICR triggered by Ca 2+ influx through VDCC to E-C coupling in SMCs have been defined in highly excitable SMCs such as VD and urinary bladder (6,7,9). In contrast, it has been suggested that the [Ca 2+ ] i rise due to Ca 2+ influx through L-type VDCC during an action potential may possibly be large enough to elicit a twitch contraction in some types of SMCs (32).…”

Methyl-β-cyclodextrin Prevents Ca2+-Induced Ca2+ Release in Smooth Muscle Cells of Mouse Urinary Bladder

“…The black dots showed membrane currents under whole cell voltage-clamp. ] i then slowly spread from the spots to the entire intracellular area, forming a Ca 2+ wave even after repolarization (13). In contrast, in MβCD-pretreated myocytes, Ca 2+ hot spots appeared more slowly and less extension of the Ca 2+ wave was observed.…”

“…A large outward current followed the initial inward current (Ca 2+ current). Most of the outward current recorded at 0 mV (by over 80%) was inhibited by addition of 1 µM paxilline or 100 nM iberiotoxin (not shown), indicating that a large part of the outward current is the BK channel current (11,13). The activation of outward current appeared to be slower in MβCD-pretreated myocytes.…”

“…The functional coupling between VDCC and RyR in the junctions, which are presumably formed by caveolae and superficial SR, may be reduced by the MβCD-induced collapse of caveolae. In the previous study, we have reported that Ca 2+ release during E-C coupling in UBSMCs occurs in two-steps (13 It has been reported that the pre-treatment with MβCD rather increases BK channel current upon depolarization in human myometrium SMCs in the presence of 1 mM EGTA in the pipette solution (6). This phenomenon has been interpreted as follows: BK channels in caveolae may form an actin-channel-caveolin complex and the destruction of the complex increases BK channel activity.…”

Ca2+ entry following P2X receptor activation induces IP3 receptor‐mediated Ca2+ release in myocytes from small renal arteries

“…In some phasic smooth muscles, for example, urinary bladder and vas deferens, Ca 2+ entry through VGCCs is tightly coupled to RyR-mediated Ca 2+ release. Electrical stimulation of these SMCs, in which close proximity of VGCCs and RyRs within the caveolar domains was confirmed with 3D-immunofluorescence and electron microscopy (Moore et al, 2004), triggers an abrupt RyR-mediated Ca 2+ release at multiple sub-PM regions ('hot spots ', Ohi et al, 2001;Morimura et al, 2006). The ability of Ca 2+ entering the SMC via VGCCs to induce RyR-mediated Ca 2+ release has also been demonstrated in voltage-clamp experiments performed on SMCs from pregnant myometrium (Shmigol et al, 1998), ileum (Kohda et al, 1997), mesenteric artery (Bolton and Gordienko, 1998), cerebral arteries (Kamishima and McCarron, 1997) and portal vein (Coussin et al, 2000).…”