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.

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

2001 ◽  
Vol 281 (2) ◽  
pp. C555-C562 ◽  
Author(s):  
Madhumita Jena Mohanty ◽  
Maian Ye ◽  
Xingli Li ◽  
Noreen F. Rossi
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Nicotinic Acid ◽  
Vascular Smooth Muscle Cells ◽  
Ruthenium Red ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Hypotonic Swelling

Hypotonic swelling increases the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). The source of this Ca2+ is not clear. To study the source of increase in [Ca2+]i in response to hypotonic swelling, we measured [Ca2+]i in fura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a 40.7-nM increase in [Ca2+]i that was not inhibited by EGTA but was inhibited by 1 μM thapsigargin. Prior depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increase in [Ca2+]i in response to hypotonic swelling. Exposure of 45Ca2+-loaded intracellular stores to hypotonic swelling in permeabilized VSMC produced an increase in45Ca2+ efflux, which was inhibited by 1 μM thapsigargin but not by 50 μg/ml heparin, 50 μM ruthenium red, or 25 μM thio-NADP. Thus hypotonic swelling of VSMC causes a release of Ca2+ from the intracellular stores from a novel site distinct from the IP3-, ryanodine-, and nicotinic acid adenine dinucleotide phosphate-sensitive stores.

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.

Ruthenium red selectively prevents Ins(1,4,5)P3-but not caffeine-gated calcium release in avian atrium

1992 ◽  
Vol 262 (1) ◽  
pp. H268-H277 ◽  
Author(s):  
A. M. Vites ◽  
A. J. Pappano
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Release ◽  
Present Report ◽  
Ruthenium Red ◽  
Detectable Effect ◽  
Calcium Accumulation ◽  
Two Agents ◽  
Release Mechanisms ◽  
Subsequent Response ◽  
Inositol 1,4,5 Trisphosphate

We previously reported that inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and caffeine evoked contractures in saponin-permeabilized chick atria. The magnitude of contractures evoked by maximally effective concentrations of Ins(1,4,5)P3 were half those evoked by maximally effective concentrations of caffeine. In the present report, we tested the hypothesis that these two agents may act on distinct calcium-release mechanisms by comparing the effects of ryanodine, ruthenium red, and procaine on the responses to Ins(1,4,5)P3 and caffeine. We find that procaine inhibits both responses with similar mean inhibitory concentrations in the millimolar range. Nanomolar concentrations of ryanodine selectively potentiate the contractures induced by Ins(1,4,5)P3 but have no effect on those induced by caffeine. Micromolar concentrations of ryanodine inhibit responses to both Ins(1,4,5)P3 and caffeine in a use-dependent way. Ruthenium red prevents the response to Ins(1,4,5)P3 and potentiates that to caffeine, as if ruthenium red had enhanced calcium accumulation in the caffeine-sensitive pool(s). Because we found that caffeine prevented the subsequent response to Ins(1,4,5)P3, but Ins(1,4,5)P3 had no detectable effect on the caffeine-induced contracture, we conclude that Ins(1,4,5)P3 and caffeine act on pharmacologically distinct calcium-release mechanisms that may reside in the same sarcoplasmic reticulum compartment.

scholarly journals Mfge8 attenuates human gastric antrum smooth muscle contractions

2020 ◽  
Author(s):  
Wen Li ◽  
Ashley Olseen ◽  
Yeming Xie ◽  
Cristina Alexandru ◽  
Brian A. Perrino
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Milk Fat ◽  
Smooth Muscles ◽  
Smooth Muscle Contraction ◽  
Gastric Antrum ◽  
Mechanical Responses ◽  
Gastric Smooth Muscle ◽  
Milk Fat Globule ◽  
Smooth Muscle Contractions

AbstractCoordinated gastric smooth muscle contraction is critical for proper digestion and is adversely affected by a number of gastric motility disorders. In this study we report that the secreted protein Mfge8 (milk fat globule-EGF factor 8) inhibits the contractile responses of human gastric antrum muscles to cholinergic stimuli by reducing the inhibitory phosphorylation of the MYPT1 (myosin phosphatase-targeting subunit 1) subunit of MLCP (myosin light chain phosphatase), resulting in reduced LC20 (smooth muscle myosin regulatory light chain 2) phosphorylation. We show that endogenous Mfge8 is bound to its receptor, α8β1 integrin, in human gastric antrum muscles, suggesting that human gastric antrum muscle mechanical responses are regulated by Mfge8. The regulation of gastric antrum smooth muscles by Mfge8 and α8 integrin functions as a brake on gastric antrum mechanical activities. Further studies of the role of Mfge8 and α8 integrin in regulating gastric antrum function will likely reveal additional novel aspects of gastric smooth muscle motility mechanisms.

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.

Stretch-induced Ca2+ release via an IP3-insensitive Ca2+ channel

2002 ◽  
Vol 283 (2) ◽  
pp. C456-C462 ◽  
Author(s):  
Madhumita Jena Mohanty ◽  
Xingli Li
Keyword(s):  
Smooth Muscle ◽  
Nicotinic Acid ◽  
Vascular Smooth Muscle Cells ◽  
Mechanical Stimulation ◽  
Ruthenium Red ◽  
Cytochalasin D ◽  
Mechanical Stimuli ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Cyclical Stretch

Various mechanical stimuli increase the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). A part of the increase in [Ca2+]i is due to the release of Ca2+ from intracellular stores. We have investigated the effect of mechanical stimulation produced by cyclical stretch on the release of Ca2+ from the intracellular stores. Permeabilized VSMC loaded with45Ca2+ were subjected to 7.5% average (15% maximal) cyclical stretch. This resulted in an increase in45Ca2+ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP3), ryanodine, and nicotinic acid adenine dinucleotide phosphate channels (NAADP) with 50 μg/ml heparin, 50 μM ruthenium red, and 25 μM thio-NADP, respectively, did not block the increase in45Ca2+ efflux in response to cyclical stretch. However, 10 μM lanthanum, 10 μM gadolinium, and 10 μM cytochalasin D but not 10 μM nocodazole inhibited the increase in45Ca2+ efflux. This supports the existence of a novel stretch-sensitive intracellular Ca2+ store in VSMC that is distinct from the IP3-, ryanodine-, and NAADP-sensitive stores.

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.

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

1998 ◽  
Vol 274 (5) ◽  
pp. C1199-C1205 ◽  
Author(s):  
Karnam S. Murthy ◽  
Gabriel M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Muscle Cells ◽  
Ruthenium Red ◽  
Dependent Protein Kinase ◽  
Cell Content ◽  
Gastric Smooth Muscle ◽  
Dependent Protein ◽  
Permeabilized Muscle

Recent studies on the role of nitric oxide (NO) in gastrointestinal smooth muscle have raised the possibility that NO-stimulated cGMP could, in the absence of cGMP-dependent protein kinase (PKG) activity, act as a Ca2+-mobilizing messenger [K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 ( Gastrointest. Liver Physiol. 28): G660–G671, 1993]. This notion was examined in dispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) and with NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 μM), NO (1 μM), and VIP (1 μM) stimulated45Ca2+release (21 ± 3 to 30 ± 1% decrease in45Ca2+cell content); Ca2+ release stimulated by 8-BrcGMP was concentration dependent with an EC50 of 0.4 ± 0.1 μM and a threshold of 10 nM. 8-BrcGMP and NO increased cytosolic free Ca2+ concentration ([Ca2+]i) and induced contraction; both responses were abolished after Ca2+ stores were depleted with thapsigargin. With VIP, which normally increases [Ca2+]iby stimulating Ca2+ influx, treatment with PKA and PKG inhibitors caused a further increase in [Ca2+]ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release nor contraction induced by cGMP and NO in permeabilized muscle cells was affected by heparin or ruthenium red. Ca2+ release induced by maximally effective concentrations of cGMP and inositol 1,4,5-trisphosphate (IP3) was additive, independent of which agent was applied first. We conclude that, in the absence of PKA and PKG activity, cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.

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