Inositol 1,4,5-trisphosphate receptors modulate  Ca2+ sparks and Ca2+ store content  in vas deferens myocytes

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 μM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 μM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in ∼20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 μM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 μM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 μM), which sensitizes the IP3R to IP3. In cells voltage clamped at –40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

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The Influence of Sarcoplasmic Reticulum Ca2+ Concentration on Ca2+ Sparks and Spontaneous Transient Outward Currents in Single Smooth Muscle Cells

The Journal of General Physiology ◽

10.1085/jgp.113.2.215 ◽

1999 ◽

Vol 113 (2) ◽

pp. 215-228 ◽

Cited By ~ 119

Author(s):

Ronghua ZhuGe ◽

Richard A. Tuft ◽

Kevin E. Fogarty ◽

Karl Bellve ◽

Fredric S. Fay ◽

...

Keyword(s):

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Potassium Channels ◽

Smooth Muscle Cells ◽

High Speed ◽

Muscle Cells ◽

Wide Field ◽

Outward Currents ◽

The Relationship ◽

Spontaneous Transient Outward Currents

Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca2+-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentration within the sarcoplasmic reticulum and a Ca2+ spark and that between a Ca2+ spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the sarcoplasmic reticulum ([Ca2+]SR) and in the cytosol ([Ca2+]CYTO) along with STOCs. At a holding potential of 0 mV, cells displayed Ca2+ sparks and STOCs. Ca2+ sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca2+]CYTO at the time and location of the spark peak was ∼100 nM above a resting concentration of ∼100 nM. The frequency and amplitude of spontaneous Ca2+ sparks recorded at −80 mV were unchanged for a period of 10 min after removal of extracellular Ca2+ (nominally Ca2+-free solution with 50 μM EGTA), indicating that Ca2+ influx is not necessary for Ca2+sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca2+]SR in association with a surge in [Ca2+]CYTO and a fusion of STOCs, followed by a fast restoration of [Ca2+]CYTO and a gradual recovery of [Ca2+]SR and STOCs. The return of global [Ca2+]CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca2+. After the global [Ca2+]CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca2+]SR, even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca2+]SR was restored to 60% or more of resting levels. At [Ca2+]SR levels above 80% of rest, there was a steep relationship between [Ca2+]SR and STOC frequency. In contrast, the relationship between [Ca2+]SR and STOC amplitude was linear. The relationship between [Ca2+]SR and the frequency and amplitude was the same for Ca2+ sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca2+]SR might provide one mechanism whereby agents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca2+ release site and the Ca2+-activated potassium channels responsible for a STOC.

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Surface Features of Smooth Muscle Cells from the Mesenteric Artery and Vas Deferens

Journal of Cell Science ◽

10.1242/jcs.8.2.427 ◽

1971 ◽

Vol 8 (2) ◽

pp. 427-443 ◽

Cited By ~ 1

Author(s):

C. E. DEVINE ◽

F. O. SIMPSON ◽

W. S. BERTAUD

Keyword(s):

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Guinea Pig ◽

Smooth Muscle Cells ◽

Mesenteric Artery ◽

Vas Deferens ◽

Muscle Cells ◽

Mesenteric Arteries ◽

Dense Bodies ◽

Freeze Etching

Smooth muscle cells of small mesenteric arteries and vas deferens of guinea-pig were examined by freeze-etching. The most striking finding was that the surface vesicles lie in roughly longitudinal rows, with areas of membrane free of vesicles in between. The areas free of vesicles are believed to correspond to areas occupied by dense bodies in conventionally fixed and sectioned material. Other cell constituents which could be identified included sarcoplasmic reticulum and, probably, thick myofilaments.

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