Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

1995 ◽  
Vol 151 (2) ◽  
pp. 149-155 ◽  
Author(s):  
A. B. Elmoselhi ◽  
M. Blennerhassett ◽  
S. E. Samson ◽  
A. K. Grover
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Skinned Smooth Muscle

Primary action of endothelin on Ca release in bovine coronary artery smooth muscle cells

1991 ◽  
Vol 260 (4) ◽  
pp. C763-C770 ◽  
Author(s):  
C. Wagner-Mann ◽  
L. Bowman ◽  
M. Sturek
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Physiological Solution ◽  
Prolonged Treatment ◽  
Free Solution ◽  
Ca Channels ◽  
Voltage Gated ◽  
Primary Action ◽  
Sustained Increase

Intracellular free Ca concentrations (Cai) were determined by fura-2 microfluorometry in single freshly dispersed cells to differentiate endothelin (ET)-induced Ca release from Ca influx through voltage-gated Ca channels (VGCC). In physiological solution ET (10(-8) M) significantly (P less than 0.05) increased Cai 23 +/- 3% (+/- SE) above baseline; this increase was not significantly attenuated by 2 x 10(-4) M lanthanum, a blocker of VGCC, or Ca-free solution. When the sarcoplasmic reticulum was depleted of Ca by prolonged treatment with 5 x 10(-3) M caffeine, depolarization with 80 mM K (80K; or 30K) plus ET did not increase Cai above that induced by 80K (or 30K) in caffeine alone. In contrast, 10(-6) M BAY K 8644, instead of ET in the protocol, significantly (P less than 0.05) increased Cai above that induced by 80K (or 30K). ET released Ca from the caffeine-sensitive internal store but was not rapid and transient like caffeine-induced release, which elicited a peak Cai increase in less than 1 min; instead, release was more gradual and prolonged with Cai peaking in greater than 2 min, thus resembling the response to 10(-5) M ryanodine. With two ET exposures, either a transient nonrepeatable increase in Cai or a delayed, but sustained, increase in Cai resulted, similar to the response to ryanodine. These data indicate that in freshly dispersed bovine cells the predominant mechanism by which ET increases Cai is release of Ca from the sarcoplasmic reticulum; if any increase in L-type voltage-gated Ca influx occurred, it was minimal and matched by efflux.

Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle

2006 ◽  
Vol 290 (3) ◽  
pp. H1136-H1144 ◽  
Author(s):  
Xiu-Yu Yi ◽  
Victoria X. Li ◽  
Fan Zhang ◽  
Fan Yi ◽  
Daniel R. Matson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Vascular Function ◽  
Hplc Analysis ◽  
Spectrometric Analysis ◽  
Channel Activity ◽  
Open Probability ◽  
Maximal Increase ◽  
Diphenylene Iodonium

It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2−· in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2−· into the cytosol. It is imperative to clarify the source of intracellular O2−· associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2−· locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2−·, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 ± 0.0005 to 0.0213 ± 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2−·.

Peroxide inactivates calcium pumps in pig coronary artery

1992 ◽  
Vol 263 (2) ◽  
pp. H537-H543 ◽  
Author(s):  
A. K. Grover ◽  
S. E. Samson ◽  
V. P. Fomin
Keyword(s):  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Dual Effect ◽  
Partial Protection ◽  
Calcium Pumps ◽  
Pig Coronary Artery

To study the effects of hydrogen peroxide, pig coronary artery smooth muscle subcellular fractions enriched in plasma membrane (F2) or sarcoplasmic reticulum (F3) were incubated in various concentrations of peroxide and 5 mM azide. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ uptake was determined for F3 and phosphate-stimulated uptake for F2. Only 1.5-5 microM hydrogen peroxide was required for 50% inhibition of the Ca2+ uptake by F3, but the corresponding concentration for F2 was 10-50 microM. This effect was not prevented by superoxide dismutase. Hydrogen peroxide inhibited the Ca(2+)-dependent formation of a 115-kDa acylphosphate band in F3 and 140- and 115-kDa bands in F2. The inhibition of Ca2+ uptake in F3, however, exceeded the inhibition of the acylphosphate formation. Efflux of Ca2+ from F2 and F3 was enhanced by hydrogen peroxide but F3 was more sensitive than F2. We conclude that hydrogen peroxide has dual effect on Ca2+ dynamics in the coronary artery smooth muscle, i.e., it inactivates the Ca2+ pumps and increases membrane permeability to Ca2+. The effect is more pronounced on sarcoplasmic reticulum than on plasma membrane. Intrinsic catalase may, however, provide partial protection against such damage.

Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle

2003 ◽  
Vol 284 (2) ◽  
pp. C294-C301 ◽  
Author(s):  
Ashok K. Grover ◽  
Sue E. Samson ◽  
Sarah Robinson ◽  
Chiu Yin Kwan
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Quaternary Structure ◽  
Ischemia Reperfusion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Vesicular Membrane ◽  
Cardiac Muscles ◽  
Pig Coronary Artery

Peroxynitrite generated in arteries from superoxide and NO may damage Ca2+ pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca2+ into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca2+ uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca2+ pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca2+ uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca2+ pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.

Sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle is regulated by a novel pathway

1996 ◽  
Vol 271 (1) ◽  
pp. C181-C187 ◽  
Author(s):  
A. K. Grover ◽  
A. Xu ◽  
S. E. Samson ◽  
N. Narayanan
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Phosphorylation Site ◽  
Cyclopiazonic Acid ◽  
Western Blots ◽  
Calmodulin Antagonist ◽  
Calmodulin Kinase ◽  
Kinase Phosphorylation

Coronary artery smooth muscle expresses an alternative splice (SERCA2b) of the sarcoplasmic reticulum (SR) Ca2+ pump gene SERCA2, which is also expressed in cardiac muscle (SERCA2a), but how the activity of this transporter is regulated in the coronary artery is not known. SERCA2a in the cardiac muscle can be regulated via phospholamban or, as recently reported, by a direct phosphorylation of this protein by calmodulin kinase (Xu, A., C. Hawkins, and N. Narayanan. J.Biol. Chem. 268:8394-8397, 1993). Because both SERCA2a and SERCA2b contain this calmodulin kinase phosphorylation site, we examined the effect of endogenous calmodulin kinase phosphorylation of the SR Ca2+ pump in the coronary artery. SR-enriched membranes were isolated from coronary artery smooth muscle and washed in ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to remove bound calmodulin. When these membranes were incubated with MgATP2- in the presence of Ca2+/calmodulin, a 115-kDa protein was phosphorylated. This phosphorylated 115-kDa protein was identified as SERCA2b in Western blots and by immunoprecipitation using a SERCA2-selective antibody. Preincubating the membranes in MgATP2- in the presence of Ca2+/calmodulin stimulated the subsequent Ca2+ uptake in the presence of oxalate plus MgATP2- and azide. The stimulation of Ca2+ uptake was inhibited by including the SR Ca2+ pump inhibitors thapsigargin and cyclopiazonic acid in the Ca2+ uptake medium or by including the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or the calmodulin kinase II peptide fragment 290-309 in the phosphorylation solution. Thus an endogenous calmodulin-dependent kinase phosphorylated SERCA2b and activated it. Phospholamban could not be detected in these membranes in Western blots. Therefore, the regulation of the SR Ca2+ pump activity in coronary artery smooth muscle may involve a direct phosphorylation of the pump protein by an endogenous calmodulin-dependent kinase.

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