Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle

1994 ◽  
Vol 15 (6) ◽  
pp. 682-700 ◽  
Author(s):  
Graeme F. Nixon ◽  
Gregory A. Mignery ◽  
Avril V. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Immunogold Localization ◽  
Tonic Smooth Muscle ◽  
Inositol 1,4,5 Trisphosphate

Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

1992 ◽  
Vol 262 (5) ◽  
pp. C1258-C1265 ◽  
Author(s):  
Y. T. Xuan ◽  
O. L. Wang ◽  
A. R. Whorton
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.

Ca2+ stores in smooth muscle cells: Ca2+ buffering and coupling to AVP-evoked inositol phosphate synthesis

1994 ◽  
Vol 266 (1) ◽  
pp. C276-C283 ◽  
Author(s):  
D. M. Berman ◽  
T. Sugiyama ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Inverse Relationship ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Enzyme System ◽  
Similar Treatment ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Communication Pathway

Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while decreasing the Ca2+ content of sarcoplasmic reticulum (SR) stores in cultured A7r5 cells at rest and after activation with 8-arginine vasopressin (AVP). Decreasing Ca2+ influx by reducing extracellular Ca2+ or by treatment with verapamil had no effect on resting [Ca2+]cyt but significantly inhibited the AVP-evoked Ca2+ transients (delta Ca2+). Neither treatment affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Likewise, basal [3H]InsP were unaffected by brief (10-30 s) exposures to thapsigargin (TG), while AVP-induced [3H]InsP synthesis was significantly augmented. Similar treatment with TG rapidly increased resting [Ca2+]cyt and decreased SR Ca2+ by 9-25% as manifested by decreased delta Ca2+. By contrast, ryanodine induced slow increases in [Ca2+]cyt that stabilized within 30 min; subsequent AVP-induced delta Ca2+ were attenuated by 50%. Ryanodine had no effect on either basal or stimulated [3H]InsP levels. Agents that elevate adenosine 3',5'-cyclic monophosphate (cAMP) such as caffeine, 8-bromo-cAMP, and forskolin inhibited AVP-evoked [3H]InsP formation. These observations provide further characterization of a communication pathway between the AVP-sensitive Ca2+ stores in the SR and the plasmalemmal enzyme system involved in the synthesis of inositol 1,4,5-trisphosphate. This pathway is manifested by an inverse relationship between the Ca2+ content of an AVP-sensitive, ryanodine-insensitive SR Ca2+ store and evoked [3H]InsP synthesis and may represent an important component in the tonic regulation of resting [Ca2+]cyt and vasoconstrictor- and hormone-evoked SR Ca2+ release.

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

2003 ◽  
Vol 285 (1) ◽  
pp. C195-C204 ◽  
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.

Nitric oxide inhibits calcium release from sarcoplasmic reticulum of porcine tracheal smooth muscle cells

1997 ◽  
Vol 272 (1) ◽  
pp. L1-L7 ◽  
Author(s):  
M. S. Kannan ◽  
Y. S. Prakash ◽  
D. E. Johnson ◽  
G. C. Sieck
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Tracheal Smooth Muscle ◽  
Nitric Oxide Donor ◽  
Ip3 Receptors ◽  
Video Fluorescence Microscopy ◽  
Inositol 1,4,5 Trisphosphate

In the present study, effects of the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), on sarcoplasmic reticulum (SR) Ca2+ release were examined in freshly dissociated porcine tracheal smooth muscle (TSM) cells. Fura 2-loaded TSM cells were imaged using video fluorescence microscopy. SR Ca2+ release was induced by acetylcholine (ACh), which acts principally through inositol 1,4,5-trisphosphate (IP3) receptors, and by caffeine, which acts principally through ryanodine receptors (RyR). SNAP inhibited ACh-induced SR Ca2+ release at both 0 and 2.5 mM extracellular Ca2+. Degraded SNAP had no effect on ACh-induced SR Ca2+ release. SNAP also inhibited caffeine-induced SR Ca2+ release. ACh-induced Ca2+ influx was not affected by SNAP when SR reloading was blocked by thapsigargin. SNAP also did not affect SR Ca2+ reuptake. The membrane-permeant analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromo-cGMP, mimicked the effects of SNAP. These results suggest that, in porcine TSM cells, SNAP reduces the intracellular Ca2+ response to ACh and caffeine by inhibiting SR Ca2+ release through both IP3 and RyR, but not by inhibiting influx or repletion of the SR Ca2+ stores. These effects are likely mediated via cGMP-dependent mechanisms.

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

2002 ◽  
Vol 80 (6) ◽  
pp. 588-603 ◽  
Author(s):  
Marguerite A Stout ◽  
Luc Raeymaekers ◽  
Humbert De Smedt ◽  
Rik Casteels
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Slow Component ◽  
Main Pulmonary Artery ◽  
Adenosine Diphosphate ◽  
Ruthenium Red ◽  
Gastric Antrum ◽  
Gastric Smooth Muscle ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cyclic Adenosine Diphosphate

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15–33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 μM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca+2 store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.Key words: calcium channels, smooth muscle, sarcoplasmic reticulum.

Intracellular Cl− fluxes play a novel role in Ca2+ handling in airway smooth muscle

2006 ◽  
Vol 290 (6) ◽  
pp. L1146-L1153 ◽  
Author(s):  
Simon Hirota ◽  
Nancy Trimble ◽  
Evi Pertens ◽  
Luke J. Janssen
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Benzoic Acid ◽  
Airway Smooth Muscle ◽  
Channel Blocker ◽  
Negative Charge ◽  
Ryanodine Receptors ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Uptake And Release

Intracellular Ca2+ is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca2+ from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca2+ across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca2+ into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl− (Cl[Formula: see text]) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca2+. Thus inhibition of SR Cl− fluxes will reduce Ca2+ sequestration and agonist-induced release. The Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10−4 M), previously shown to inhibit SR Cl− channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a “run down” of sequestered Ca2+ within the SR. Niflumic acid (10−4 M), a structurally different Cl− channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca2+ concentration ([Ca2+]i). Depletion of Cl[Formula: see text], accomplished by bathing ASM strips in Cl−-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl− depletion significantly reduced caffeine-induced increases in [Ca2+]i. Together these data suggest a novel role for Cl[Formula: see text] fluxes in Ca2+ handling in smooth muscle. Because the release of sequestered Ca2+ is the predominate source of Ca2+ for contraction of ASM, targeting Cl[Formula: see text] fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.

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