Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

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The effects of acetylcholine and atropine on the 22Na+ permeability of intestinal smooth muscle vesicles

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y78-146 ◽

1978 ◽

Vol 56 (6) ◽

pp. 921-925

Author(s):

L. Spero

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Intestinal Smooth Muscle ◽

Erythrocyte Ghosts ◽

Muscarinic Agonists ◽

Plasma Membrane Vesicles ◽

Muscle Plasma Membrane ◽

Whole Muscle ◽

Kinetics Of

A technique is described which has enabled us to measure changes in 22Na+ efflux from smooth muscle plasma membrane vesicles. The resting 22Na+ efflux from these sealed vesicles showed a concentration-dependent increase in response to acetylcholine and other muscarinic agonists, in similar concentrations to those which increased 42K+ efflux in whole muscle. The kinetics of this efflux were complex and could not be described by less than three exponential processes. The response to agonists has, therefore, been characterized by measurement of the half-life of 22Na+ efflux (t1/2). The acetylcholine effect was inhibited by atropine, but unlike the situation in the whole muscle, this inhibition was noncompetitive. Tubocuraine (a nicotinic antagonist) had no effect on this acetylcholine response. Atropine has no effect by itself on the resting 22Na+ efflux, neither did tetrodotoxin or ouabain. 22Na+ efflux from erythrocyte ghosts and liposomes, prepared from lipid extracts of the smooth muscle plasma membrane, was not modified by acetylcholine or atropine.

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Preferential support of Ca 2+ uptake in smooth muscle plasma membrane vesicles by an endogenous glycolytic cascade

The FASEB Journal ◽

10.1096/fasebj.3.11.2528493 ◽

1989 ◽

Vol 3 (11) ◽

pp. 2298-2301 ◽

Cited By ~ 66

Author(s):

Richard J. Paul ◽

Christopher D. Hardin ◽

Luc Raeymaekers ◽

Frank Wuytack ◽

Rik Casteels

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Muscle Plasma Membrane

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Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium

Biochemical Journal ◽

10.1042/bj2520215 ◽

1988 ◽

Vol 252 (1) ◽

pp. 215-220 ◽

Cited By ~ 27

Author(s):

A Enyedi ◽

J Minami ◽

A J Caride ◽

J T Penniston

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Calcium Uptake ◽

Limited Proteolysis ◽

Membrane Vesicles ◽

Calcium Pump ◽

Ionophore A23187 ◽

Plasma Membrane Vesicles ◽

Active Calcium ◽

Stimulation Of

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.

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Calcium pump, high-affinity Ca2+-ATPase, and other ATPases in dog antrum smooth muscle plasma membrane

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.5.c449 ◽

1985 ◽

Vol 248 (5) ◽

pp. C449-C456 ◽

Cited By ~ 36

Author(s):

A. K. Grover ◽

C. Y. Kwan ◽

P. J. Oakes

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Maximal Activity ◽

Calcium Pump ◽

Disulfonic Acid ◽

Calmodulin Antagonists ◽

High Affinity ◽

Chelex 100 ◽

Muscle Plasma Membrane ◽

Absolute Requirement

The plasma membrane-enriched fraction from dog antrum smooth muscle is enriched in ATP-dependent azide-insensitive Ca2+ uptake (0.3-0.4 microM Ca2+ required for half-maximal activity), a high-affinity Ca2+-ATPase (Km of 0.3-0.8 microM for Ca2+), a low-affinity Ca2+-ATPase (Km for 250-400 microM for Ca2+), and a Mg2+-ATPase. Studies using membranes washed with EDTA and assay media treated with Chelex 100 showed that the high-affinity Ca2+-ATPase did not depend on contaminating Mg2+. Thus, whereas the ATP-dependent Ca2+ uptake had an absolute requirement for Mg2+, the Ca2+-ATPases did not. Studies using gamma-irradiation showed that the protein responsible for the ATP-dependent Ca2+ uptake was inactivated at significantly lower doses of radiation than the three ATPases. The Ca2+ uptake and the high-affinity Ca2+-ATPase also differed in their inhibition by calmodulin antagonists and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus it is unlikely that the high-affinity Ca2+-ATPase by itself is responsible for the ATP-dependent Ca2+ uptake.

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ATP-dependent calcium pump and Na+-Ca2+ exchange in plasma membrane vesicles from squid optic nerve

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(86)90557-2 ◽

1986 ◽

Vol 860 (3) ◽

pp. 583-591 ◽

Cited By ~ 5

Author(s):

Luis Osses ◽

Madalina Condrescu ◽

Reinaldo DiPolo

Keyword(s):

Plasma Membrane ◽

Optic Nerve ◽

Membrane Vesicles ◽

Calcium Pump ◽

Plasma Membrane Vesicles

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Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi

Biochemical Journal ◽

10.1042/bj3060299 ◽

1995 ◽

Vol 306 (1) ◽

pp. 299-303 ◽

Cited By ~ 21

Author(s):

G Benaim ◽

S N J Moreno ◽

G Hutchinson ◽

V Cervino ◽

T Hermoso ◽

...

Keyword(s):

Plasma Membrane ◽

Trypanosoma Cruzi ◽

Mammalian Cells ◽

Membrane Vesicles ◽

High Sensitivity ◽

Bovine Brain ◽

Calcium Pump ◽

Plasma Membrane Vesicles ◽

P Type

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

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Calcium uptake by placental plasma membrane vesicles

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(75)90309-0 ◽

1975 ◽

Vol 401 (2) ◽

pp. 256-264 ◽

Cited By ~ 12

Author(s):

Y Shami ◽

H.H Messer ◽

D.H Corp

Keyword(s):

Plasma Membrane ◽

Calcium Uptake ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles

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Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium

Biochemical Journal ◽

10.1042/bj2610023 ◽

1989 ◽

Vol 261 (1) ◽

pp. 23-28 ◽

Cited By ~ 7

Author(s):

A Enyedi ◽

J Brandt ◽

J Minami ◽

J T Penniston

Keyword(s):

Plasma Membrane ◽

Sarcoplasmic Reticulum ◽

Inhibitory Action ◽

Calcium Uptake ◽

Membrane Vesicles ◽

Young Female ◽

Female Rats ◽

Maximal Velocity ◽

Percoll Gradient ◽

Plasma Membrane Vesicles

Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboestrol was administered together with progesterone, the inhibitory action of oxytocin was totally suppressed, consistent with the expected action of this agent. The results suggest that regulation of the plasma membrane Ca2+ pump may be important in the prolonged elevation of intracellular Ca2+ caused by oxytocin.

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