Mary E. Carsten

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.

Prostaglandins and oxytocin: Their effects on uterine smooth muscle*

Abstract A subcellular fraction was isolated from uteri of non-pregnant and pregnant cows. ATP-dependent calcium binding was shown to take place in this fraction. This calcium binding was inhibited in a dose related fashion when increasing amounts of prostaglandin (PG) E2 or F2α were added to the in vitro experimental medium. The physiologically inactive PGF1β had no inhibitory effect. Oxytocin caused inhibition of calcium binding in preparations from both pregnant and non-pregnant cows. The response to PGE2 and PGF2α was somewhat greater in preparations from pregnant uteri than from non-pregnant uteri. The response to oxytocin was very much greater in pregnant uteri. Because of the high PG sensitivity of calcium binding in preparations from the non-pregnant uterus, it is concluded that the PGs may be the more suitable agent in the control of reproduction.

ACTIN: A COMPARATIVE STUDY.

Abstract Actin preparations from striated muscle of beef, pig, lamb, chicken, frog, fish and pecten were compared with respect to their sedimentation coefficients, starch-gel electrophoresis, amino acid composition and peptide maps. No differences were found in the actin preparations from mammalian and avian muscle. When compared to mammalian actin, chemical differences were noted in the case of frog, fish and pecte actin. Actin from frog muscle differed only in the peptide map while the fish actin differed in amino acid composition and peptide map. The most pronounced differences appeard in the pecten actin which differed in starch-gel electrophoresis pattern, amino acid composition and peptide map. The results thus demonstrate structural differences between actins from species over a wide range of the evolutionary scale; the differences decreased with closer relationship of the specied.

Prostaglandins and cellular calcium transport in the pregnant human uterus

The attempt was made to shed some light on the cellular mechanism of prostaglandin action in the human myometrium. Focus was on calcium transport in the human pregnant uterus as affected by prostaglandins. A subcellular tissue fraction was isolated from human pregnant uteri by differential centrifugation and purified on a sucrose density gradient. This fraction bound calcium in the presence of adenosine triphosphate (ATP), splitting ATP in the process. The preparations were characterized both morphologically and chemically. The observed activity seemed to be similar to ATP-dependent calcium-binding activity in sarcoplasmic reticulum (SR) isolated from striated muscles. The smooth muscle stimulator prostaglandin E1 inhibited ATP-dependent calcium binding and enhanced calcium release from SR preparations. Physiologically inactive prostaglandin F1beta had no effect on SR calcium binding. With the demonstration of calcium-binding system in uterine smooth muscle, there is little question that the mechanism which starts uterine muscle contraction is the release of calcium from the membrane system associated in muscle cells with the SR. In sum, there is the temptation to suggest that the onset of contractions in the human uterus and possibly the activity which culminates in labor are regulated by endogenous or exogenous prostaglandins.

Hormonal Regulation of Myometrial Calcium Transport

Purified preparations of fragmented sarcoplasmic reticulum from non-pregnant bovine uterus bind calcium from a medium containing ATP and Mg++. This calcium binding was enhanced in the presence of progesterone, while it is decreased in the presence of oxytocin. In the presence of both hormones there was an intermediate effect. PGF2Α, when added to preparations that had previously bound calcium in the presence of progesterone, caused release of limited amounts of calcium. Under these conditions calcium binding was much higher than that observed in the presence of PGF2Α alone. Three steroid analogs showed no effect on ATP-dependent calcium binding.

Uterine smooth muscle: Troponin☆

Abstract Similarity in the mechanism of contraction in smooth and skeletal muscle is based on the presence of contractile proteins, similar or identical in structure, properties and biological activity, in both types of muscle. Troponin, which together with tropomyosin confers calcium sensitivity to actomyosin, is shown to be present in bovine uterine smooth muscle. Uterine troponin was purified by isoelectric precipitation and ammonium sulfate fractionation. The uterine troponin preparations were shown to be free of tropomyosin in acrylamide gel electrophoresis. Electrophoresis in sodium dodecyl sulfate showed a minimum of six components. The intrinsic viscosity of uterine troponin was greatly increased upon addition of uterine tropomyosin indicating complex formation of the two proteins. The biological activity of uterine troponin was similar to that of skeletal troponin. The uterine troponin-tropomyosin complex inhibited uterine and skeletal actomyosin ATPase in the presence of EGTA.

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.

Cardiac Sarcotubular Vesicles: EFFECTS OF IONS, OUABAIN AND ACETYLSTROPHANTHIDIN

The transport of calcium through the membranes of the sarcoplasmic reticulum plays an important role in the relaxation-contraction cycle of the heart. To study and characterize this system, fragmented sarcotubular vesicles from dog hearts were prepared by ultracentrifugation. The fraction between 15,000 and 40,000 g was partially purified by gradient centrifugation. Calcium accumulation by the vesicles in the presence of ATP, Mg, 45Ca, and oxalate was studied. The rate of calcium uptake increased with the ATP concentration and was not affected by changes in K or Na concentration nor by substitution of isosmotic amounts of sucrose for 75% of the Na ions. Splitting of ATP was increased upon substitution of sucrose. In aged preparations of vesicles, ouabain or acetylstrophanthidin decreased the rate of their calcium accumulation and partially reversed the inhibition of myofibrillar ATPase by the vesicles. It is suggested that the partial inhibition of calcium uptake increases the concentraton of free calcium, and thus makes more calcium available for contraction of the myofibril.

Actin from Heart Muscle: Studies on Amino Acid Composition

The sedimentation behavior, patterns on starch gel electrophoresis, amino acid compositions, and peptide maps following tryptic digestion of dog heart and skeletal actin preparations have been compared. The results from all methods failed to demonistrate differences between the actin preparations, suggesting a very high degree of similarity, if not identity, between these contractile proteins from heart and skeletal muscle.