Calcium/calmodulin-dependent kinase 2 mediates Epac-induced spontaneous transient outward currents in rat vascular smooth muscle

Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKIIδ2 with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with γ-tubulin. Furthermore, CaMKIIδ2 coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca2+-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKIIδ2 mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIδ2 using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKIIδ2 may be an important regulator of iNOS trafficking and activity in VSMCs.

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Spontaneous transient outward currents in smooth muscle cells

Cell Calcium ◽

10.1016/s0143-4160(96)90103-7 ◽

1996 ◽

Vol 20 (2) ◽

pp. 141-152 ◽

Cited By ~ 101

Author(s):

T.B. Bolton ◽

Y. Imaizumi

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Outward Currents ◽

Spontaneous Transient Outward Currents

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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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Oxygen-induced fetal pulmonary vasodilation is mediated by intracellular calcium activation of KCa channels

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.6.l1379 ◽

2001 ◽

Vol 281 (6) ◽

pp. L1379-L1385 ◽

Cited By ~ 26

Author(s):

Valerie A. Porter ◽

Michael T. Rhodes ◽

Helen L. Reeve ◽

David N. Cornfield

Keyword(s):

Smooth Muscle ◽

Pulmonary Artery ◽

Smooth Muscle Cells ◽

Intracellular Calcium ◽

Muscle Cells ◽

Pulmonary Vasodilation ◽

Outward Currents ◽

Acute Increase ◽

Pulmonary Artery Smooth Muscle ◽

Spontaneous Transient Outward Currents

O2 sensing in fetal pulmonary artery smooth muscle is critically important in the successful transition to air breathing at birth. However, the mechanism by which the fetal pulmonary vasculature senses and responds to an acute increase in O2tension is not known. Isolated fetal pulmonary artery smooth muscle cells were kept in primary culture for 5–14 days in a hypoxic environment (20–30 mmHg). These cells showed a 25.1 ± 1.7% decrease in intracellular calcium in response to an acute increase in O2 tension. Low concentrations of caffeine (0.5 mM) and diltiazem also decreased intracellular calcium. The decrease in intracellular calcium concentration in response to increasing O2 was inhibited by iberiotoxin and ryanodine. Freshly isolated fetal pulmonary artery smooth muscle cells exhibited “spontaneous transient outward currents,” indicative of intracellular calcium spark activation of calcium-sensitive potassium channels. The frequency of spontaneous transient outward currents increased when O2 tension was increased to normoxic levels. Increasing fetal pulmonary O2 tension in acutely instrumented fetal sheep increased fetal pulmonary blood flow. Ryanodine attenuated O2-induced pulmonary vasodilation. This study demonstrates that fetal pulmonary vascular smooth muscle cells are capable of responding to an acute increase in O2tension and that this O2 response is mediated by intracellular calcium activation of calcium-sensitive potassium channels.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00374.2002 ◽

2003 ◽

Vol 285 (1) ◽

pp. C195-C204 ◽

Cited By ~ 33

Author(s):

Carl White ◽

J. Graham McGeown

Keyword(s):

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Guinea Pig ◽

Vas Deferens ◽

Confocal Imaging ◽

Time Duration ◽

Outward Currents ◽

Inositol 1,4,5 Trisphosphate ◽

Dependent Pathway ◽

Spontaneous Transient Outward Currents

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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RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle

The Journal of General Physiology ◽

10.1085/jgp.200308999 ◽

2004 ◽

Vol 123 (4) ◽

pp. 377-386 ◽

Cited By ~ 53

Author(s):

Guangju Ji ◽

Morris E. Feldman ◽

Kai Su Greene ◽

Vincenzo Sorrentino ◽

Hong-Bo Xin ◽

...

Keyword(s):

Smooth Muscle ◽

Calcium Release ◽

Ryanodine Receptors ◽

Wild Type ◽

Calcium Dependent ◽

Outward Currents ◽

Associated Proteins ◽

Kinetics Of ◽

Spontaneous Transient Outward Currents

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

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