Jordan D. Miller

Ca2+ Release by inositol trisphosphate from Ca2+-transporting microsomes derived from uterine sarcoplasmic reticulum

Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 microM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that by mobilizing intracellular Ca2+, inositol trisphosphate may be the link between hormonal stimuli and smooth muscle contraction.

A new look at uterine muscle contraction

Recent progress in our understanding of uterine smooth muscle contraction is reviewed. We no longer believe that actin-myosin interaction in the myometrium occurs through activation of the thin filament; but it is triggered by calcium-dependent phosphorylation of myosin in the thick filament. Calcium is now thought to originate from both extracellular and intracellular sources. Calcium can enter the cell through either a voltage- or a hormone-controlled calcium channel. The intracellular source of calcium is the sarcoplasmic reticulum. The effect of oxytocin in human labor is no longer considered the result of increased circulating oxytocin but rather of increased oxytocin receptors. In contrast, the contractile action of some prostaglandins is related to increased prostaglandin formation at human parturition. The step between hormone binding and cellular action is mediated by second messengers. The uterine-relaxing action of cyclic adenosine monophosphate is now thought to be limited to the inhibition of myosin phosphorylation. Recently discovered second messengers for contraction of the myometrium are phosphoinositides; their turnover causes calcium release from the sarcoplasmic reticulum. Guanine nucleotides are thought to be modulators of these two second messengers.

Prostaglandin E2 receptor in the myometrium: Distribution in subcellular fractions

Abstract [ 3 H]Prostaglandin (PG) E 2 bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE 2 and PGE 1 competed for the [ 3 H]PGE 2 binding site. The PGs inhibited in the following decreasing order: PGE 2 = PGE 1 ⪢ PGF 2α > PGA 2 > PGF 1β > PGB 2 . No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10 −9 . Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10 −11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10 −11 mol binding sites/g. The results suggest that there is a high-affinity PGE 2 receptor present in both plasma membrane and sarcoplasmic reticulum.

Comparison of calcium association constants and ionophoretic properties of some prostaglandins and ionophores

Abstract The prostaglandin calcium association constants and calcium transport rates are reported. The calcium association constants for prostaglandins B 2 and E 2 were similar to one another, but lower than those of the ionophores A23187 and X537A. Using a Pressman cell, the ionophores A23187 and X537A, as well as prostaglandin B 2 , were found to transport calcium through an organic phase, while the prostaglandin E 2 calcium transport rate was not appreciable in the artifical system.

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Abstract Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. 125I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE2) binding was found to be a property of the cell membrane.

Calcium-loaded microsomes from uterine smooth muscle. A biological system to test for calcium ionophore action of prostaglandins.

Smooth muscle microsomal vesicles were loaded with calcium in the presence of oxalate and ATP. The intact vesicles that contained calcium oxalate crystals were separated by ultracentrifugation from empty vesicles. This results in a unique model system, composed of a biologically active membrane with virtually all of the calcium inside. Intravesicular calcium is differentiated from externally bound calcium using ionophore X537A and EGTA. EGTA released calcium slowly with a half time of 93 min. The ionophore X537A rapidly released calcium with a half time of 12 min. This model can be used to test for calcium ionophoretic action. Prostaglandin (PG) E2 and prostacyclin (PGI2) were tested in this system. We found that PGE2 and PGI2 did not change calcium permeability.

Properties of a phosphorylated intermediate of the Ca,Mg-activated ATPase of microsomal vesicles from uterine smooth muscle

A phosphorylated intermediate of the CaMg-ATPase is demonstrated in microsomal preparations from uterine smooth muscle. Characterization included the use of activators, inhibitors, and sodium dodecyl sulfate (SDS)-gel electrophoresis. The phosphorylation was a function of the ATP and Ca concentrations. The dissociation constant KATP was 2.7 X 10(-6) M and KCa was 1.7 X 10(-6) M. Mg was obligatory for the reaction. Na azide, ouabain, or the substitution of NaCl for KCl did not affect the reaction. Phosphorylation was inhibited by Salyrgan, ADP, or 20 mM calcium. SDS-polyacrylamide gel electrophoresis at pH 2.4 demonstrated phosphorylation of predominantly one protein with a molecular weight of 100,000. Hydroxylamine and, to a lesser extent, neutral and alkaline pH caused dephosphorylation. This indicates the presence of an acylphosphate bond in the phosphoprotein. The above findings are consistent with the phosphorylated intermediate being a Ca,Mg-ATPase. The inhibition by 20 mM calcium indicates that the Ca,Mg-ATPase of smooth muscle differs from that of striated muscle sarcoplasmic reticulum.

Calcium Control Mechanisms in the Myometrial Cell and the Role of the Phosphoinositide Cycle

Smooth muscle contraction follows excitation at the plasma membrane. The link between these two events is the rise in intracellular calcium. Calcium can enter through plasma membrane channels activated either by depolarization of the plasma membrane or by specific binding of a hormone to its receptor. Hormone—receptor binding can be followed by the breakdown of plasma membrane phosphoinositides with the generation of a series of messengers. These serve multiple functions, including the release of calcium from intracellular stores into the cytoplasm. In this chapter we first discuss the biochemical reactions that cause changes in intracellular calcium concentration. This is followed by a review of the phosphoinositide system and its role in the release of second messengers that initiate myometrial contraction.

The binding of Ca2+ to Ca2+-transporting microsomes derived from bovine uterine sarcoplasmic reticulum

An obligatory early step in the transport of calcium across the internal membranes of smooth muscle cells is the binding of calcium to the Ca,Mg-ATPase. The characterization of calcium binding to sarcoplasmic reticulum from smooth muscle has not been reported. Calcium binding to a bovine myometrium preparation was investigated using Scatchard analysis and a computer program utilizing weighted least squares curve fitting and an exact mathematical model of binding. This permitted objective measurement of goodness of fit and showed that best fit was obtained using a two site model. Magnesium did not change the affinity for calcium of the two sites; but reduced the number of low affinity sites to half.