A new look at uterine muscle contraction

Current Development

A new look at uterine muscle contraction Mary E. Carsten, Ph.D., and Jordan D. Miller, M.D. Los Angeles, California 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. Calciurn 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. (AM J OssTET GYNECOL 1987;157:1303-15.)

The past decade has seen the following three new developments contribute to our understanding of smooth muscle contraction and in particular to uterine muscle contraction: (I) The actin-myosin interaction in uterine smooth muscle was shown to be different from that in skeletal muscle; (2) the dominant role of intracellular calcium in smooth muscle contraction was firmly established; and (3) a new second messenger, inositol trisphosphate, was discovered. The common denominator for these reactions is calcium. In this review, recent findings will be discussed, and molecular mechanisms will be reviewed as far as they are known. Since not all experimental data are available for the myometrium, at times we will have to refer to other smooth muscles. This is with some misgivings because where detailed studies have been done, differences have been found, even between arteries from various locations. Nevertheless, we have made progress in our understanding of normal events in myometrial contraction.

Contraction Smooth muscle structure. While it had been known for many years that actin and myosin were present in smooth muscle, it was thought that smooth muscle con-

From the Departments of Obstetrics and Gynecology and Anesthesiology, School of Medicine, University of California, Los Anr;eles. Reprint requests: Mary E. Carsten, Ph.D., Department of Obstetrics and Gynecology, UCLA Medical School, Rehabilitation Center. Room A5-38, Los Angeles, CA 90024-1789-22.

traction was similar to that of skeletal muscle, which is represented by the sliding filament model. Arrays of thick protein filaments containing the protein myosin interdigitate with arrays of thin filaments containing the protein actin. In contraction the filaments move past each other because of actin-myosin interaction without a change in length of the individual filament, but the muscle as a whole changes its length. Interaction of actin and myosin occurs through the myosin crossbridges, protrusions from the myosin filament that reversibly bind to actin. The energy for contraction comes from adenosine triphosphate (ATP) hydrolysis. In skeletal muscle the interaction of actin and myosin is inhibited by the presence of the tropomyosin-troponin complex in the thin filament. The inhibition is removed when troponin binds calcium, which causes a steric shift of the complex. Attempts to visualize thick filaments in smooth muscle were unsuccessful for many years. This was partly from technical difficulties and partly from the lower myosin content of smooth muscle. 1 In smooth muscle, the alignment of the filaments is less regular; hence no cross-striations are observed. The thin filaments can be arranged in parallel with the cell axis, in arrays of various shapes, and in rosettes when in association with myosin, or at random. 2· ' The actin filaments connect through dense bodies with the plasma membrane. This modification allows for many features characteristic of smooth muscle-its ability to shorten to less than 50% and lengthen to more than twice its resting length, greater force, and low velocity of contraction.

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November 1987 Am J Obstet Gynecol

y

ca++

Calm

ATP~Ki~ADP

Ca• C a l m y MLCK'

MLCK-P

Myosin<

>

-Po s r · Actomyosin-P My

ADP

Phospha.!ase

Actin

1

ATP

ATP~ADP

Actin

Relaxation

Contraction

Fig. 1. Diagram of contraction-relaxation cycle of smooth muscle. (Calm = Calmodulin; cAMP kinase = cAMP-dependent protein kinase; MLCK = myosin light-chain kinase; MLCK-P = phosphorylated myosin light-chain kinase; Myosin-P = phosphorylated myosin; Actomyosin-? = phosphorylated actomyosin; ADP = adenosinediphosphate; ATP = adenosine triphosphate.)

The actin-myosin interaction in smooth muscle. In smooth muscle, most of the evidence indicates that troponin plays only a minor role, if any, in the contractionrelaxation cycle. Myosin consists of four poiypeptide chains, two heavy chains and two light chains. In smooth muscle contraction, the 20,000 molecular weight myosin light chain is phosphorylated by the enzyme myosin light-chain kinase when the calcium concentration rises. Only when phosphoryiated can the myosin light chain interact with actin. The reaction requires the protein calmodulin; the calcium-calmodulin complex then binds to myosin light-chain kinase for activation. When the calcium level decreases, the calmodulin dissociates from the myosin light-chain kinase and the myosin light-chain kinase becomes inactive. Dephosphorylation of the myosin light chain occurs at a steady rate by a phosphatase.•· 5 This reaction has been demonstrated in vitro and in intact smooth musde. 6 The activity of the myosin light-chain kinase is modulated by a cyclic adenosine monophosphate (cAMP)-dependent protein kinase. This protein kinase catalyzes phosphorylation of myosin light-chain kinase.7 The phosphorylated myosin light-chain kinase has only a weak affinity for the calcium calmodulin complex, thus becoming inactive. Dephosphorylation of myosin and relaxation follow. 6 • 8 This reaction sequence is illustrated in Fig. 1. In the myometrium, myosin light-chain phosphorylation and dephosphorylation were demonstrated for the contraction-relaxation cycle. 9 Serine and threonine residues of the myosin light chain were phosphorylated.10 Less popular hypotheses, which may well play a role as auxiliary mechanisms for smooth muscle contraction, focus on calcium stimulation acting on the thin filament. One hypothesis involves the binding of the

protein leiotonin to actin 11 , the other, the calciumdependent regulation by the protein caldesmon, present in the thin filaments. 2 No attempts have been made so far to demonstrate these proteins in the myometrium. Pharmacomechanical coupling

The events leading to muscle contraction start at the cell membrane with a stimulus. The stimulus initiates ari action potential, which is followed by a contraction. This is called electromechanical coupling and is a means of excitation-contraction coupling in skeletal muscle. It has been found that in smooth muscle, electromechanical coupling does not fully explain observed phenomena. 12 In smooth muscle, there is not always an action potential. There can be either electrical or hormonal stimulation. Hormone binding to a receptor at the cell surface can activate a chain of events that ultimately lead to muscle contraction. This is called pharmacomechanical coupling. 13 The search for its mechanism and some of the answers are discussed in the following sections. The role of calcium

The smooth muscle cell membrane. A diagram of the smooth muscle cell is shown in Fig. 2, and the actin arid myosin filaments are seen. At 10-s to 10- 7 M calcium, the muscle is relaxed, and at 10- 6 M calcium, the filaments interact and the muscle contracts without changing filament length. From where does the calcium for contraction come? It has long been known that part of the calcium comes from outside the cell. The calcium concentration outside the cell in the interstitial fluid is 10- 3 M, that is, 10,000-fold higher than that inside the cell. Although there is an electrochemical gradient, calcium does not rush into the cell at rest because of the

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10-3 M Ca ++

Ca

++

1305

voc

Fig. 2. Calcium sources for myometrial contraction. On top, sliding filament of muscle contraction (left) and relaxation (right). (Ac = Actin; Tm = tropomyosin; My = myosin; P- = phosphorylated; SR = sarcoplasmic reticulum; R = receptor; Ag = agonist; ROC = receptm·-operated channel; VOC = voltage-operated channel; ATP = adenosine triphosphate; ADP = adenosine diphosphate.)

low calcium permeability of the cell membrane. Calcium can enter the cell through an ion channel with an action potential. Calcium binds reversibly to a specific site at or near the surface of the channel. Subsequent movement of the calcium ion is determined by current flow and the extracellular concentration of calcium. Calcium channels. Some chemical agents that inhibit contraction have been found to directly block calcium entry. They are called calcium channel blockers, or calcium entry blockers; they bind to the cell membrane within the membrane calcium channel.'' They make up a wide range of structures ranging from inorganic ions such as lanthanum to some organic compounds. The more interesting ones are the dihydropyridines, nitrendipine, nifedipine, nicardipine, and other unrelated compounds such as diltiazem, verapamil, and D 600, Some of the latter have been found to have additional action in the myometrium.'" Observations on muscle strips from the parturient rat uterus showed that D 600 and other calcium entry blockers abolished spontaneous contractions and reduced the force of electrically evoked contractions. However, D 600 did not block the tonic contraction evoked by prostaglandin (PG) F2a.' 6 · ' 7 These experiments suggested the presence of another type of calcium channel, namely, one controlled by hormones. It was subsequently shown in rabbit aorta that the amount of calcium influx on maximal stimulation by potassium or norepinephrine was dissimilar and each was less than the calcium influx when both were applied simultaneously. This led to the suggestion that an additional set of calcium channels could be opened by norepinephrine after maximal calcium influx caused by potassium.' 8 Furthermore, D 600 pref-

erentially inhibited potassium-activated channels, and in general, higher concentrations of calcium entry blockers are required to block the receptor-operated channel than the voltage-operated channel. Clinically, calcium channel blockers produce adequate relaxation and have been used to inhibit spontaneous contractions in premature labor' 9 · 2 ' and myometrial hyperactivity in primary dysmenorrhea. 22•24 Calcium efflux through the cell membrane. The calcium that entered the cell for contraction must eventually be pumped out of the celL The energy requiring transport of calcium across membranes is mediated by enzymes, the so-called calcium and magnesium adenosine triphosphatases (Ca,Mg-ATPase), because these enzymes split adenosine triphosphate (ATP) and require calcium and magnesium for their activation. The ATPase of the cell membrane is stimulated by the protein calmodulin 25 and inhibited by the chemical vanadate. 26 In cardiac muscle an important mechanism for moving calcium across the cell membrane is N a+ -Ca + + exchange. This mechanism wotks by removing calcium from the inside of the cell in exchange for incoming sodium. The Na+-Ca++ exchange has been observed in some smooth muscles including the uterus. 27 ·"'0 However, the Na + -Ca + + exchange appears to be of low magnitude in smooth muscle, which may be from technical difficulties in experimentation. Its physiologic significance is debated. 18 Role of the sarcoplasmic reticulum. Recent observations have shown that the influx of calcium with an action potential is insufficient to activate fully the contractile mechanism and sustain maximum contraction

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of smooth muscle. 31 Much of the required calcium comes from intracellular stores, but there are quantitative differences between various smooth muscles. In skeletal muscle there is an abundant system of intracellular channels or tubules, called the sarcoplasmic reticulum. This system stores calcium in relaxation. On an impulse conducted from the cell membrane, this calcium is rapidly released during contraction. This mechanism is useful because of the large size of the skeletal muscle cell and the speed of contraction. In contrast, the smooth muscle cell is much smaller than the skeletal muscle cell and contractions are slow. Hence the role of the sarcoplasmic reticulum in smooth muscle contraction was questioned. Early observations demonstrated a less abundant sarcoplasmic reticulum in smooth muscle cells.' 2 · '" However, recent studies with the electron microscope have shown a sarcoplasmic reticulum similar to that in skeletal muscle." The sarcoplasmic reticulum is estimated to occupy about 1.5% to 7.5% of the cell volume of the smooth muscle cell and is particularly abundant in some blood vessels and in pregnant or estrogen-treated uterus. 31 It has been demonstrated that in vascular smooth muscle, in calciumfree solution, and in the presence of lanthanum, which blocks passage of calcium through the cell membrane, intracellular stores of calcium are sufficient to elicit maximal contraction through recycling of intracellular calcium stores. 35 Ultrastructural studies that use electron-probe analysis have shown high calcium concentrations in the sarcoplasmic reticulum of relaxed smooth muscles. From these findings it was concluded that the sarcoplasmic reticulum and not the cell membrane is the organelle primarily responsible for the physiologic regulation of the cytoplasmic calcium. 31 This hypothesis is also borne out by physiologic and biochemical observations. It is presently thought that mitochondria play no role in the regulation of cytoplasmic calcium at physiologic levels. 37 In relaxation calcium is sequestered by the sarcoplasmic reticulum. This mechanism for reducing the intracellular calcium concentration is in addition to the extrusion of calcium through the plasma membrane previously discussed. Calcium uptake into the sarcoplasmic reticulum. Calcium uptake into the sarcoplasmic reticulum will be discussed first. More is known about calcium uptake than about calcium release, since the former has been easier to study. The transport of calcium into the sarcoplasmic reticulum can best be demonstrated on the subcellular level in microsomal preparations. The techniques for isolating microsomal preparations are based on the procedures used for the preparation of the sarcoplasmic reticulum of skeletal muscle. They were modified because of the large amount of connective tissue in smooth muscle that prevents easy isolation of smooth muscle components. Furthermore, smooth muscle con-

November 1987 Am .J Obstet Gynecol

tains much more cell membrane than does skeletal muscle because of the smaller size of the smooth muscle cell. Thus it is harder to prepare pure sarcoplasmic reticulum from the smooth muscle cell. The degree of purity of the preparation is difficult to document because there are no unique markers.'"· 3" In our laboratory we have isolated sarcoplasmic reticulum from term pregnant bovine myometrium and human uteri obtained at cesarean hysterectomy.1('.' 2 Under the electron microscope our sarcoplasmic reticulum preparation shows vesicles of about 1000 A (100 nm) diameter."' In the presence of ATP and calcium, the sarcoplasmic reticulum preparations accumulate calcium on the inside of the vesicles, that is, they take up calcium from solution. The calcium uptake increases with an increased amount of calcium in solution'' and is stimulated by oxalate. The process is temperaturedependent and ATP is required. This in vitro process of calcium removal from solution and transport into a storage area would cause in vivo muscle relaxation. Adenosine triphosphatase. Parallel with calcium uptake is ATPase activity. A Ca,Mg-ATPase in the sarcoplasmic reticulum membrane breaks down ATP to adenosine diphosphate (ADP) by removing the terminal phosphate group.' 2 This Ca,Mg-ATPase has different characteristics from the ATPase of the cell membrane. These characteristics serve to identify the origin of the isolated microsomal preparations. In contrast to the ATPase of the cell membrane, the ATPase of the sarcoplasmic reticulum is not sensitive to stimulation by calmodulin or inhibition by vanadate. 2 '· 2 " 11 On the molecular level the ATPases of sarcoplasmic reticulum and cell membrane differ in molecular weight (sarcoplasmic reticulum 100,000 to 110,000; cell membrane 130,000) as shown by electrophoresis of the phosphor" ylated intermediate." "' With tryptic digestion, little difference could be found between the molecular weight of the ATPase of aortic smooth muscle and that of skeletal muscle. However, there are immunologic differences. ' 7 The tryptic digestion pattern of the ATPase of the sarcoplasmic reticulum is different from that of the cell membrane ATPase. 17 Mechanism of calcium transport. To understand the events that occur in the transport of calcium through the membranes of the sarcoplasmic reticulum, it is advantageous to examine partial reactions, since these can be more easily interpreted. Several steps in the cycle of calcium uptake into sarcoplasmic reticulum vesicles and calcium release and the mechanism of action of the Ca,Mg-ATPase have been recently elucidated. The first step in calcium transport is the binding of calcium to the outside of the vesicular membrane of sarcoplasmic reticulum. Two types of calcium binding sites were found with association constants of 8 and 0.1 (X 106 M-'). The number of binding sites was four and 10 nrriol/mg of protein, respectively. The high-affinity binding site appears to be part of the transport ATPase;

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the presence of a second binding site may indicate that calcium serves an additional function!" The use of magnesium sulfate as a tocolytic agent in the treatment of premature labor49 -51 is well known. It has been shown that Mg+ + abolishes contractions in uterine muscle strips. 52 In the presence of 5 mM Mg+ +, the maximum number of low-affinity calcium sites was reduced to half, whereas the high-affinity calcium binding sites were not affected by Mg+ +.18 Whether the changes observed in vitro correlate with the in vivo action of Mg+ + remains to be documented. The next step in calcium transport is the formation of a high-energy intermediate in the presence of ATP and calcium. This is demonstrated by showing a phosphorylated ATPase. Using [32 P]ATP, we obtained results that document phosphorylation of the ATPase," which were consistent with the following scheme, modeled after that presently established for calcium transport in skeletal muscle. Ca""'

+E+

+ ADP + P + Ca;..

ATP~Ca · E · P

Ca · E ·

P~E

where E is the 110,000 molecular weight ATPase enzyme. E binds calcium plus ATP with activation of the enzyme. The formation of an intermediate, Ca · E · P, results from the binding of a terminal phosphate, P, of ATP to the aspartyl residue of the ATPase. The ATP is split, ADP is released, calcium is then transported to the inside of the vesicles, and phosphate is released. Calcium release. The reverse process, the release of calcium from the sarcoplasmic reticulum vesicles, does not require energy and leads in vivo to contraction. Experiments with microsomal vesicles have shown that the vesicles are relatively impermeable to calcium with a half time of 100 minutes at 37" C. This means that it will take I 00 minutes for half the accumulated calcium to diffuse out of the vesicles. However, this can be speeded up by use of an ionophore. Ionophores move calcium through a membrane down the concentration gradient. The release of calcium with an inophore shows that the calcium is inside the vesicles 53 ; however, this is not a physiologic mechanism. Therefore we are left with the question, what is the physiologic mechanism for calcium release? Hormonal calcium mobilization. The uptake and release of calcium can be inhibited or enhanced by various agents. As shown in partially purified microsomal preparations in our laboratory, the uterine contractile prostaglandins E 2 (PGE 2 ) and F 2 " (PGF 2 .) inhibited calcium uptake and enhanced calcium release; oxytocin also inhibited calcium uptake. Consistent with its pharmacologic action, PGE 2 was more potent than PGF 2". Much greater effects of oxytocin and slightly greater effects of PGF 2 • were observed in microsomes derived from pregnant uteri than in those from nonpregnant uteri. 41 · 42 • 54 · 55 The increase in free calcium is a pre-

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requisite for contraction. Similar effects of PGF 2• were recently observed in vascular smooth muscle cells. 5" The release of calcium from intracellular calcium stores by prostaglandins is further supported by experiments showing prostaglandin-induced contraction of rat uterine horns in calcium-free medium. 57 It is unclear whether this is a direct effect of prostaglandins or whether it is mediated through a messenger. Hormonal stimulation and hormone receptors

The two physiologic hormones that cause uterine contractions are oxytocin and prostaglandin. Oxytocin is the preferred pharmacologic agent for induction of labor because of its high specificity in contracting the uterus. However, the physiologic role of these two hormones in the onset of human labor is controversial, and the picture is muddled by great differences between species. In the context of this discussion, it is unnecessary to go into arguments of which is the more important. Oxytocin. Some workers have shown a rising trend in oxytocin concentration in the maternal plasma with gestation. This trend increased toward the end of pregnancy.5" 59 However, technical difficulties with the assay, fluctuating patterns, and wide scatter of obtained oxytocin values make these results questionable. More recent work showed no rise in plasma oxytocin concentration until late in the second stage of labor.""· 61 An alternative explanation for the effect of oxytocin would be a rise in oxytocin receptors. This is manifested by increased sensitivity to oxytocin. Increased sensitivity of the myometrium to oxytocin with advancing pregnancy, especially toward the very end of pregnancy, has long been known. 62 Indeed, it was shown that the sensitivity to oxytocin correlated with preterm and postterm delivery, with women who delivered prematurely having the highest sensitivity to oxytocin. 63 Specific receptors for oxytocin have been identified in the rat, 64 · 65 ewe, 66 rabbit, 5 7 and human uterus.""· 69 In binding experiments with purified subcellular membrane fractions, the oxytocin receptors were found to be located in the myometrial cell membrane. The dissociation constant was calculated to be in the range of 2 to 3 nM."'· 66 · 67 The concentration of oxytocin receptors rises toward the end of pregnancy, is maximal during labor, and then declines."' In subcellular preparations from a human uterus, there was also an increase in oxytocin receptor concentration with gestational age, as well as some increase in binding affinity at term."" Similar changes in oxytocin receptor concentration were found in human uteri at various stages of gestation and in labor, with lower values before the onset oflabor and in failed induction of labor with oxytocin. 69 Furthermore, the concentration of oxytocin receptors appears to be regulated by estrogen and progesterone, at least in the rat and rabbit, with estrogen increasing the number of oxytocin receptors and progesterone inhib-

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iting this increase. 64 · 7"· 71 These findings correlate with observed physiologic function and suggest that the increase in uterine sensitivity to oxylocin is caused by an increase in myometrial oxytocin binding sites. Prostaglandins. Unlike oxytocin, which is a circulating hormone, prostaglandins originate at or near their site of action, that is, in the endometrial and myometrial cell. Therefore one would expect differences in the physiologic action of these two hormones. Early work indicated that administration of PGE 2 and PGF 2• and prostaglandin analogs by various routes causes contraction of the pregnant and nonpregnant uterus and can initiate labor or abortion at any stage of gestation. n. 73 Prostaglandins play an important role in both the nonpregnant and pregnant uterus as evidenced by physiologic, pathologic, and pharmacologic findings. During the menstrual cycle, high concentrations of both PGE and PGF were found in the human myometrium during the late proliferative phase, coinciding with ovulation. After ovulation the concentration of PGE gradually declined and stayed low, whereas the PGF concentration showed a second peak during the late secretory phase, approximately coinciding with the onset of menstruation. 71 However, in humans and primates, prostaglandins have no luteolytic effect, in contrast to domestic animals. 75 76 High levels of PGF 2 • and PGE were found to be present in menstrual fluid collected from women suffering from primary dysmenorrhea. 77 Prostaglandin synthesis inhibitors can stop 78 menstrual cramps and pain in primary dysmenorrhea. Prostaglandins are detected by radioimmunoassay in the amniotic fluid at about midpregnancy, and their concentration rises through the remainder of pregnancy and into spontaneous labor, with PGE 2 levels higher than those of PGF 2 •• 79 Their concentration in the amniotic fluid, as well as that of arachidonic acid, are higher during labor than before labor and increase with labor. 80 The levels of PGF were found to be higher in women going into spontaneous labor than in women requiring oxytocin induction. 8 L 82 However, measurement of prostaglandins in the amniotic fluid is influenced by the mode of obtaining the samples; results are higher in samples collected at amniotomy than in those obtained by amniocentesis. 83 The measurement of prostaglandins in peripheral plasma is even more difficult than that in amniotic fluid because of their low concentration, the ability of blood platelets to synthesize prostaglandins, and the destruction of prostaglandins by one passage through the lungs. 84 More reliable measurements are obtained by monitoring a prostaglandin metabolite, 13, 14-dihydro15-keto-PGF, which is present in much higher concentration and not synthesized in platelets. With these measurements no increase in prostaglandin concentration in the peripheral blood was noted up to the onset of labor. 85 On the other hand, 13,14-dihydro-15-keto-PGF

November 1987 Am J Obstet Gynecol

levels have been found to rise concomitant with cervical ripening without uterine activity before the onset of labor. 86 Taken together these findings lead to the concept that increased synthesis of prostaglandins plays a dominant role in human parturition. The events that lead to the formation of prostaglandins in human parturition begin in the fetal membranes (amnion and chorion) and also in the decidua. Prostaglandin production is greatest in the amnion, followed by the chorion, and finally the decidua. 87 The first step is the release of free arachidonic acid. Arachidonic acid is the obligatory precursor of the prostaglandins of the "2" series. Arachidonic acid is not stored in human tissues, but human fetal membranes accumulate arachidonic acid in esterified form in the form of glycerophospholipids. The major pathway of arachidonic acid release is by phospholipase A 2 action. This enzyme breaks down glycerophospholipids in the "2" position. The end products of hydrolysis are free fatty acid and lysoglycerophospholipid. Lysoglycerophospholipids are cytolytic agents. They break down cell membranes and also lysosomal membranes inside the cell. Lysosomes contain more phospholipase. Its release causes accelerated release of arachidonic acid and thus accelerated prostaglandin synthesis from preformed glycerophospholipids in the fetal membranes. The preferred substrate for phospholipase A 2 in the fetal membranes is phosphatidyl ethanolamine, which has arachidonic acid in the "2" position. 80 · 88 · 8 " PGE 2 and PGF 2• are subsequently formed from arachidonic acid in a multistep process, the first step of which is catalyzed by the enzyme cyclooxygenase. 84 The intermediate endoperoxide and thromboxane also contract the uterus. A secondary pathway of arachidonic acid mobilization starts from phosphatidylinositol. Its breakdown requires three enzymes, phospholipase C, diacylglycerol lipase, and monoacylglycerollipase. 89 · 90 The stimulus for increased prostaglandin formation in the fetal membranes at the onset of human parturition may be estrogens. 91 Estradiol was shown to stimulate cyclooxygenase activity in the rat uterus 92 · 93 and aortic smooth muscle cells. 94 Stimulation of prostaglandin synthesis by oxytocin has been observed in decidua parietalis but not in decidua vera from the membranes or the myometrium. 95 · 96 There is some evidence that calcium plays a role in arachidonic acid mobilization followed by prostaglandin production in amnion tissue. 89 There is a striking association between premature labor and intrauterine, endocervical, and urinary tract infection. It has been shown that many of the organisms that cause these infections have phospholipase A 2 activity.97 This would lead to increased release of arachidonic acid from preformed glycerophospholipids in the fetal membranes, followed by prostaglandin biosynthesis and preterm labor. Inhibitors of prostaglandin synthesis may delay par·

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PI

1309

PIP

PA

'tf_

<1

~~ l

DG

OH

Arachidonic acid

l

Eioosanoids Fig. 3. Pathway of phosphatidylinositide metabolism. (PI "' Phosphatidylinositol; PIP = phosphatidylinositol 4-phosphate; PIP 2 = phosphatidylinositol 4,5-bisphosphate; IP, = inositol trisphosphate; sFA = saturated fatty acid; AA = arachidonic acid; PA = phosphatidic acid; DC = diacylglycerol.) Eicosanoids comprise prostaglandins, thromboxane, and leukotrienes.

1 turition as shown in experimental animals.'"· ' " Aspirin and indomethacin are potent inhibitors of cyclooxygenase, which catalyzes the conversion of arachidonic acid to prostaglandins. In women, prolonged intake of 1011 101 large doses of aspirin can lengthen pregnancy, · and administration of indomethacin can extend inductionabortion time in midtrimester abortion. 102 However, the use of these agents for treatment of preterm labor is disputed because they may cause premature closure of the ductus arteriosus, and aspirin, in particular, may lead to prolonged platelet dysfunction and bleeding. 111 "·HIIi PGE 2 , PGF 2• and some of their derivatives are useful tools in terminating pregnancy as in cases of an anencephalic or dead fetus 1"7 · 108 where oxytocin is ineffective. They are less suited for the initiation of labor because of their nonspecificity. Thus they may contract other smooth muscles such as those of the blood vessels and intestinal tract. This may give rise to undesirable side effects, for example, riausea, vomiting, and diarrhea. Overwhelming evidence in the literature suggests that there is interaction of prostaglandins and oxytocin and furthermore that the presence of prostaglandins is obligatory for a maximal response to oxytocin. Thus an increase in oxytocin sensitivity is prevented by ad109 ministration of prostaglandin synthetase inhibitors. myometrial In vivo, prostaglandins can lower the threshold to oxytocin. 1w In vitro, it has been shown that prostaglandins are needed for oxytocin action. Indomethacin, meclofenamate, and R-805, all potent inhibitors of prostaglandins synthetase (cyclooxygenase), antagonize the contractile effects of oxytocin but not of PGF2• on the isolated uterus of the rat and humans.111-116 Contraction of pregnant and nonpregnant

human uteri, abolished by the addition of indomethacin, was reestablished in response to PGF 2• but not to oxytocin. 113 In contrast to oxytocin, PGE and PGF 2• receptors change little in the human myometrium during pregnancy and labor. This would be in agreement with the small observed change in sensitivity to PGE through pregnancy and labor. Furthermore, there appears to be little change in prostaglandin receptors during the menstrual cycle. A single class of high-affinity binding sites was demonstrated for PGE 1with an apparent equilibrium dissociation constant (Kn) of 3.65 nM for PGE 1 and 26.9 nM for PGF 2•• 117 In purified subcellular fractions from bovine and human pregnant myometrium, prostaglandin receptors were found in the cell membrane fraction and the sarcoplasmic reticulum, which is not surprising since prostaglandins are transported by the circulation and also originate inside the cell. The affinity of cell membrane and sarcoplasmic reticulum for PGE 2 was the same. The average Kn for PGE 2 and PGE 1 was 2.50 to 3.05 nM, with the same receptor specific for both these prostaglandins. PGF2• and a series of inactive prostaglandins exhibited low competitive effects on PGE 2 binding. 118 The presence of the same receptor for PGE 1and PGE 2 but of separate receptors for PGE and PGF 2 • has been observed by many investigators.ll7-I 211 We may draw the conclusion that the physiologic changes that lead to human parturition are related to changes in the number of oxytocin receptors and to increased biosynthesis of prostaglandins. Gap junctions. In addition, gap junctions appear in the myometrium of various animals during parturition and in humans during term and preterm labor. Gap junctions are areas of low resistance to the flow of cur-

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November 1987 Am J Obstet Gynecol

/\..

Extracellular

----------,-,~rn----------------

~

PW,:A

Cell Membrane

Phospholipase C

~ ,.....-+--+

+ 1+

c.r 1+~

MLCK

Intracellular

DGc:::::::::::> Phosphatidic acid

~ Arachronic

C Kinase

acid

Prostaglandins

Fig. 4. Agonist-dependent second messengers: inositoltrisphosphate, calcium, and diacylglycerol. Agonist receptor-binding activates phospholipase C to hydrolyze phosphatidylinositol 4,5bisphosphate (PIP 2) to inositol I ,4,5-trisphosphate (IP,) and diacylglycerol (DG). MLCK "" Myosin light-chain kinase; C Kinase "" protein kinase C. MLCK and C Kinase phosphorylate various proteins, leading to biologic responses.

rent. These cell-to-cell contacts serve to couple cells and allow synchronized muscle contractions in labor. Formation of gap junctions, at least in the tat and sheep, appears to be modulated by the interaction of various hormones, such as estrogen, progesterone, and prostaglandins. 121 .123 Second messengers

Cyclic AMP. The events leading from hormone binding to hormone action are mediated by so-called second messengers. Hormones bind to specific receptors at the outside of the cell membrane. The distribution and ligand specificity of the receptors determine the tissue response and production of a second messenger. The second messenger then transposes nonspecifically to the substrate inside the cell. Adenosine-3' :5'-monophosphate (cyclic adenosine monophosphate, cAMP) has been identified as such a second messenger. Originally it was demonstrated that in the liver epinephrine releases cAMP, which in turn activates phosphorylase. 124 Based on this finding, the concept evolved that cAMP was the messenger for a multitude of other hormones. The action of cAMP is very well characterized as a messenger for epinephrine or isoproterenol, that is, for (3-adrenergic agents. The uterine-relaxing action of (3-adrenergic agents is well known. Indeed, some other relaxing agents have been shown to increase cAMP in the uterus, notably the anesthetic halothane. 125 Older theories of muscle contraction and relaxation, such as the Ying Yang theory, suggested that (3-adrenergic agents brought about relaxation through the messenger cAMP and contractile agents work through cyclic guanosine monophosphate (cyclic GMP, cGMP). 126 It was assumed that all relaxing agents work through cAMP and all contractile agents work through cGMP.

Evidence has now accumulated to show that cAMP may not be an obligatory mediator of relaxation of the uterus. This hypothesis is based on the lack of correlation between the relaxing action of hormonal or pharmacologic agents and cAMP levels in the uterus or between tension and cAMP. m. 128 Relaxation of the uterus produced by isoproterenol under various conditions in vitro does not always correlate with changes in membrane potentiaJl 29 or in tissue levels of cAMP. 1'"· 131 An explanation forwarded is that the increase in cAMP is localized and too small to be detected by chemical analysis of total tissue content of cAMP. 128 With wholeuterine muscle and a microsomal preparation from rat uterus, specific binding and cAMP production showed parallel dose-response curves as did relaxation. 132 However, it must be hypothesized that < 10% of maximal cAMP production accounts for 50% of relaxation; this has not been proved. Additional evidence for the lack of correlation of cAMP and relaxation comes from work on the role of prostaglandins in cAMP synthesis. In tissue slices PGE 1 and PGE 2 , which contract the uterus, increased cAMP levels. 133 One might argue that resul.ts were obscured because of compartmentalization. However, in our laboratory, using uterine homogenates and partially purified subcellular fractions, PGE 2 and PGF 2a, also elicited increased synthesis of cAMP, as did isoproterenol.134 The claim that cAMP enhances calcium uptake in subcellular preparations 135 · 136 was also untenable. With the use of purified microsomal preparations derived from the sarcoplasmic reticulum, 12 our laboratory showed that neither cAMP nor the protein kinase catalytic subunit increased calcium uptake. 131 Thus it appears to date that if cAMP has a relaxing action on the uterus, its mechanism is not in enhancing storage of calcium in intracellular organelles.

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More recent evidence again involves cAMP in relax-· ation. cAMP may function primarily at the level of the actin-myosin interaction (see above and Fig. 1): in the phosphorylation of the enzyme myosin light-chain kinase by the cAMP-dependent protein kinase. This inhibits myosin light-chain kinase and thus myosin lightchain phosphorylation, thereby reducing actin-myosin interaction. Cyclic GMP ..Attempts to correlate cyclic guanosine monophosphate (cGMP) levels specifically with either contraction or relaxation have failed. Thus in carbachol-induced contractions, no change in cGMP was found in the rat uterus. In guinea pig myometrium, cGMP was increased, but in carefully timed experiments, contractions were shown to precede the rise in cGMP, and no changes in cAMP were observed. 137 Furthermore, no correlation between cGMP elevation and contraction or relaxation of rat myometrium could be established. 138 Phosphoinositides. Since the nucleotide mechanism is inadequate to explain the link between hormone binding and contraction, what causes elevation of intracellular free calcium and contraction of the uterine smooth muscle cell? There is mounting evidence that binding of agonists to receptors, which leads to calcium mobilization, functions by hydrolyzing phosphoinositides. 139 · 110 The phosphoinositides are minor components of cell membranes. Their structure is shown in Fig. 3. A phosphodiesterase (phospholipase C) hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycerol and D-myoinositol-1 ,4,5-trisphosphate (inositol trisphosphate). The inositol trisphosphate acts to mobilize intracellular calcium from the endoplasmic reticulum or sarcoplasmic reticulum, as it is called in smooth muscle (Fig. 4). Diacylglycerol activates protein kinase C, which phosphorylates various proteins. 139 The inositol trisphosphate is then broken down stepwise to inositol by phosphatases which are stimulated by diacylglyceroL In the cell membrane inositol is used for stepwise resynthesis of phosphatidylinositoL These reactions have been shown in many tissues. Two experimental approaches are used to demonstrate these reactions. One is to demonstrate increased metabolites of phosphoinositides on receptor-agonist interaction; the other is to show an increase in free calcium in permeabilized cells or sarcoplasmic reticulum preparations on exposure to inositol trisphosphate. Increased hydrolysis of phosphoinositides and release of inositol trisphosphate have been observed in uterine strips on stimlllation with oxytocin or carbachoL 111 In our laboratory, addition of inositol trisphosphate caused release of calcium from isolated sarcoplasmic reticulum preparations. This calcium release was concentration dependent, and at the highest concentration used, amounted to 40% of calcium releasable by an ionophore. 112 In permeabilized cells from the mesenteric artery, the amount of calcium released by inositol

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trisphosphate was sufficient to elicit a contraction. 143 Thus one is tempted to suggest that inositol trisphosphate may be the link between agonist-receptor binding, intracellular calcium rise, and uterine smooth muscle contraction. Although there is strong evidence for the involvement of phosphoinositides in hormone-receptor binding and smooth muscle contraction, demonstration of specific inhibition of one of the enzymes involved in phosphoinositides breakdown (e.g., phospholipase C) is needed. Lack of contraction would establish the obligatory role of phosphoinositides. No specific inhibitors are available at this time. The other product in the phosphodiesterase splitting of phosphoinositide is diacylglyceroL In addition to activating protein kinase C, diacylglycerol is converted to phosphatidic acid (an ionophore) or may release arachidonic acid (Fig. 3). The latter can give rise to prostaglandin and eicosanoid biosynthesis as shown in other tissues. 90 · 144 Prostaglandins can cause release of calcium from isolated sarcoplasmic reticulum preparations of uterine smooth muscle and inhibit cakium uptake as shown in our laboratory41 · 42 · 54 ' 55 and previously discussed. Thus their action may augment the calcium release initiated by inositol trisphosphate. On the other hand, prostaglandins may actually cause breakdown of phosphoinositides after specific binding to the cell membrane; no data are available on this. Presently we suggest that accelerated synthesis of prostaglandins, stimulated by hormone-receptor binding and increased phosphoinositide metabolism, may further increase intracellular free calcium and lead to uterine contraction and possibly labor. Regulation of second messengers. Guanine nucleotides are known to be involved in modifying the cAMP system. This occurs through receptors communicating with a pair of guanine nucleotide-binding regulatory proteins (G proteins) in the cell membrane. One of these (G,) enhances the activity of adenylate cyclase, the enzyme that catalyzes cAMP synthesis, while the other (G,) inhibits. 145' 147 Guanosine 5'-triphosphate (GTP)-liganded adenylate cyclase appears to be the active species, and hydrolysis of GTP to GDP is the primary mechanism of inactivation. 118 Thus GTP is an obligatory cofactor for hormonal stimulation of adenylate cyclase. In vitro some analogs of GTP can substitute for GTP, and in binding studies that use broken cell preparations a stable analog of GTP is desirable. Particulate preparations from the smooth muscle of the rat uterus were shown to specifically bind a GTP analog with a Kn of 1.0 JJ.M. 149 Although most information about G proteins relates to the adenylate cyclase system, G proteins also appear to be mediators in the inositol trisphosphate system. It is still uncertain what type of G protein is involved. Experiments in various tissues that use permeabilized cells (mast cells) or microsomal preparations (rat liver

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or coronary artery) have shown that guanine nucleotide-binding proteins couple receptors with the hydrolysis of phosphoinositides. 150. 15" Enhancement by GTP of inositol trisphosphate stimulated calcium release has been demonstrated in rat liver, 153 but not in vascular smooth muscle cells. 151 It has also been suggested that the guanine nucleotide regulatory mechanism may operate independently in mediating calcium release from intracellular stores in nerve cells. 1"'· 157 To date no studies on the uterus have been reported. Conclusions

Recent evidence has delineated the linking of receptor activation with the production of second messengers. Hormone receptor binding at the cell membrane initiates hydrolysis of phosphoinositides to inositol trisphosphate and diacylglycerol by phospholipase C. This reaction is possibly modified by an as yet unidentified GTP-binding protein. The inositol trisphosphate diffuses to the interior of the cell and releases calcium from the sarcoplasmic reticulum. Diacylglycerol can give rise to prostaglandins, which may release more calcium from the sarcoplasmic reticulum. In the presence of calmodulin, calcium then stimulates the myosin light-chain kinase to phosphorylate myosin; contraction follows. When the calcium level declines, the myosin light-chain kinase becomes inactive. The myosin is dephosphorylated by a phosphatase. Relaxation is furthered by phosphorylation of the myosin light-chain kinase by a cAMP-dependent protein kinase. REFERENCES 1. Schoenberg CF. Contractile proteins of vertebrate smooth muscle. Nature 1965;206:526. 2. Marston SB, Smith CWJ. The thin filaments of smooth muscles. J Muscle Res Cell Motil 1985;6:669. 3. Bagby RM. Organization of contractility/cytoskeletal elements. In: Stephens NL, ed. Biochemistry of smooth muscle. Vol I. Boca Raton, Florida: CRC Press, 1983: l. 4. Haeberle JR, Hathaway DR, DePaoli-Roach AA. Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle. J Bioi Chern 1985; 260:9965. 5. Pato MD, Adelstein RS. Dephosphorylation of the 20,000-dalton light chain of myosin by two different phosphatases from smooth muscle. J Bioi Chern 1980; 255:6535. 6. Adelstein RS, Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Ann Rev Biochem 1980;49:921. 7. de Lanerolle P, Nishikawa M, Yost DA, Adelstein RS. Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science 1984;223:1415. 8. Walsh MP, Hartshorne DJ. Actomyosin of smooth muscle. In: Cheung WY, ed. Calcium and cell function. Vol III. Orlando, Florida: Academic Press, 1982:223. 9. Janis RA, Barany K, Barany M, Sarmiento JG. Association between myosin light chain phosphorylation and contraction of rat uterine smooth muscle. Malec Physiol 1981;1:3. 10. Csabina S, Mougios V, Barany M, Barany K. Characterization of the phosphorylatable myosin light chain in rat uterus. Biochim Biophys Acta 1986;871 :311.

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II. Nonomura Y, Ebashi S. Calcium regulatory mechanism in vertebrate smooth muscle. Biomed Res 1980; I: l. 12. Edman KAP, Schild HO. Calcium and the stimulant and inhibitory effects of adren<~line in depolarized smooth muscle. J Physiol 1963;169:404. 13. Somlyo AV, Somlyo AP. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J Pharmacal Exp Ther 1968;159:129. 14. Miller WC, Moore JB. High affinity binding sites for (3H)-nitrendipine in rabbit uterine smooth muscle. Life Sci 1984;34:1717. 15. Maigaard S, Forman A, Andersson K-E, Ulmsten U. Comparison of the effects of nicardipine and nifedipine on isolated human myometrium. Gynecol Obstet Invest 1983; 16:354. 16. Reiner 0, Marshall JM. Action of D-600 on spontaneous and electrically stimulated activity of the parturient rat uterus. Nauyn Schmiedebergs Arch Pharmacal 1975; 290:21. 17. Reiner 0, Marshall JM. Action of prostaglandin, PGF2" on the uterus of the pregnant rat. Nauyn Schmiedebergs Arch Pharmacal 1976;292:243. 18. van Breemen C, Aaronson P, Loutzenhiser R, Meisheri K. Ca2 + movements in smooth muscle. Chest 1980; 1:157. 19. Huddleston JF. Preterm labor. Clin Obstet Gynecol 1982;25: 123. 20. Ulmsten U, Andersson K-E, Wingerup L. Treatment of premature labor with the calcium antagonist nifedipine. Arch Gynecol 1980;229: l. 21. Csapo AI, Puri CP, Tarro S, Henzl MR. Decaptiv
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36. 37. 38.

39. 40. 41. 42.

43.

44. 45.

46.

4 7.

48.

49. 50. 51. 52.

53. 54. 55.

56.

57. 58.

in guinea-pig portal vem smooth muscle. ] Physiol· 1984;355:677. Somlyo AP, Somlyo AV, Shuman H. Electron probe analysis of vascular smooth muscle. J Cell Bioi 1979;81 :316. Somlyo AP. Cell calcium measurement with electron probe and electron energy loss analysis. Cell Calcium 1985;6: 197. Carsten ME, Miller JD. Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle. Arch Biochem Biophys 1980;204: 404. Carsten ME, Mil}er JD. Problems specific to smooth muscle fractionation. INSERM 1984; 124:293. Carsten ME. Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle.] Gen Physiol 1969;53:414. Carsten ME. Prostaglandins and cellular calcium transport in the pregnant human uterus. AM J OBSTET GY:-IECOL 1973; 117:824. Carsten ME, Miller .JD. Effects of prostaglandins and oxytocin on calcium release from a uterine microsomal fraction. A hypothesis for the ionophoretic action of prostaglandins. J Bioi Chern 1977;252:1576. Carsten ME, Miller .JD. Purification and characterization of microsomal fractions from smooth muscle. In: Casteels R, Godfraind T, Ruegg .JC, eds. Excitationcontraction coupling in smooth muscle. Amsterdam: Elsevier, 1977:155. Carsten ME, Miller JD. Modulation of phosphoprotein formation in bovine uterine microsomes. Soc Gynecol Invest 1985:216. Carsten ME, Miller .JD. Properties of a phosphorylated intermediate of the Ca,Mg-activated ATPase of microsomal vesicles from uterine smooth muscle. Arch Biochem Biophys 1984;232:616. Raeymaekers L, Wuytack F, Casteels R. Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2 +)-ATPase activity in plasmalemma and endoplasmic reticulum. Biochim Biophys Acta 1985;815:441. Chiesi M, Gasser .J, Carafoli E. Properties of the Capumping ATPase of sarcoplasmic reticulum from vascular smooth muscle. Biochem Biophys Res Commun 1984;124:797. Miller .JD, Carsten ME. The binding of Ca2+ to Ca,.transporting microsomes derived from bovine uterine sarcoplasmic reticulum. Biochem Biophys Res Commun 1987;147:13. Elliott .JP. Magnesium sulfate as a tocolytic agent. AM .J 0BSTET GYNECOL 1983;147:277. Gonik B, Creasy RK. Preterm labor: its diagnosis and management. AM .J 0BSTET GYNECOL 1986;254:3. Caritis SN, Edelstone DI, Mueller-Heubach E. Pharmacologic inhibition of preterm labor. AM .J OBSTET GYNECOL 1979;133:557. Marshall.JM. Calcium and uterine smooth muscle membrane potentials. In: Paul WM, Daniel EE, Kay CM, Monckton G, eds. Muscle. London: Pergamon Press, 1965:229. Carsten ME, Miller JD. Calcium-loaded microsomes from uterine smooth muscle. Gynecol Obstet Invest 1984; 17:78. Carsten ME. Prostaglandins and oxytocin: their effects on uterine smooth muscle. Prostaglandins 1974;5:33. Carsten ME. Can hormones regulate myometrial calcium transport? In: Stephens NL, ed. The biochemistry of smooth muscle. Baltimore: University Park Press, 1977:617. Fukuo K, Morimoto S, Koh E, et al. Effects of prostaglandins on the cytosolic free calcium concentration in vascular smooth muscle cells. Biochem Biophys Res Commun 1986;136:247. Villar A, D'Ocon D, Anselmi E. Calcium requirement of uterine contraction induced by PGE,: importance of intracellular calcium stores. Prostaglandins 1985;30:491. Dawood MY, Ylikorkala 0, Trivedi D, Fuchs F. Oxytocin

59. 60.

61. 62. 63.

64.

65. 66.

67.

68.

69.

70.

71. 72. 73.

74. 75. 76.

77. 78. 79.

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in maternal circulation and amniotic fluid during pregnancy . .J Clin Endocrinol Metab 1979;49:429. Sellers SM, Hodgson HT, Mountford LA, Mitchell MD, Anderson ABM, Turnbull AC. Is oxytocin involved in parturition? Br .J Obstet Gynaecol 1981;88:725. Leake RD, Weitzman RE, Glatz TH, Fisher DA. Plasma oxytocin concentrations in men, nonpregnant women and pregnant women before and during spontaneous labor. .J Clin Endocrinol Metab 1981;53:730. Leake RD. Oxytocin. In: MacDonald PC, Porter .J, eels. Initiation of parturition: prevention of prematurity. Columbus, Ohio: Ross Laboratories, 1983:43. Caldeyro-Barcia R, Poseiro.J.J, Theobald GW. Sensitivity of the pregnant human myometrium to oxytocin. AM .J 0BSTET GYNECOL 1968;102:1181. Takahashi K, Diamond F, Bieniarz .J, Yen H, Burd L. Uterine contractility and oxytocin sensitivity in preterm, term, and postterm pregnancy. AM .J 0BSTET GYNECOL 1980; 136:774. Alexandrova M, Soloff MS. Oxytocin receptors and parturition. I. Control of oxytocin receptor concentration in the rat myometrium at term. Endocrinology 1980; 106:730. Crankshaw DJ, Branda LA, Matlib MA, Daniel EE. Localization of the oxytocin receptor in the plasma membrane of rat myometrium. Eur .J Biochem 1978;86:481. Crankshaw DJ, Romaniuk E, Branda LA. Identification and characterization of receptors for oxytocin in the myometrium of the pregnant ewe. Gynecol Obstet Invest 1982;14:202. Riemer RK, Goldfien AC, Goldfien A, Roberts.JM. Rabbit uterine oxytocin receptors and in vitro contractile response: abrupt changes at term and the role of eicosanoids. Endocrinology 1986; 119:699. Den K, Sakamoto H, Kumura S, Takagi S. Study of oxytocin receptor: II. Gestational changes in oxytocin activity in the human myometrium. Endocrinol .J pn 1981; 28:375. Fuchs A-R, Fuchs F, Husslein P, Soloff MS, Fernstrom MJ. Oxytocin receptors and human parturition: a dual role for oxytocin in the initiation of labor. Science 1982;215:1396. Fuchs A-R, Periyasamy S, Alexandrova M, Soloff MS. Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium: effects of ovarian steroids. Endocrinology 1983; 113:742. Nissenson R, Flouret G, Hechter 0. Opposing effects of estradiol and progesterone on oxytocin receptors in rabbit uterus. Proc Nat! Acad Sci USA 1978;75:2044. Karim SMM. Physiological role of prostaglandins in the control of parturition and menstruation. J Reprod Fertil [Suppl]1972;16:105. Bygdeman M. Clinical applications. In: Samuelsson B, Ramwell PW, Paoletti R, eels. Advances in prostaglandin and thromboxane research. New York: Raven Press, 1980;6:87. Vijayakumar R, Walters WA. Myometrial prostaglandins during the human menstrual cycle. AM .J OBSTET GvNECOL 1981;141:313. Jones GS, Wentz AC. The effect of prostaglandin F2" infusion on corpus luteum function. AM .J OBSTET Gvl';ECOL 1982;114:393. Lyneham RC, Korda AR, Shutt DA, Smith ID, Shearman RP. The effect of intra-uterine prostaglandin F 2" on corpus luteum function in the human. Prostaglandins 1975;9:431. Lumsden MA, Kelly RW, Baird DT. Primary dysmenorrhoea: the importance of both prostaglandins E, and F 2". Br .J Obstet Gynaecol 1983;90: 1135. Owens PR. Prostaglandin synthetase i?hibitors in the treatment of primary dysmenorrhea. AM .J 0BSTET GYNECOL 1984; 148:96. Dray F, Frydman R. Primary prostaglandins in amniotic fluid in pregnancy and spontaneous labor. AM J Oss n: 1 GYNECO!. 1976; 126: 13.

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80. MacDonald PC, Porter JC, Schwarz BE, Johnston JM. Initiation of parturition in the human female. Semin Perinatal 1978;2:273. 81. Keirse MJNC, Flint APF, Turnbull AC. F Prostaglandins in amniotic fluid during pregnancy and labour. J OBSTET GYNAECOL Br Comm 1974;81:131. 82. Keirse MJNC, Mitchell MD, Turnbull AC. Changes in prostaglandin F and 13, 14-dihydro-15-keto-prostaglandin F concentrations in amniotic fluid at the onset of and during labour. Br J Obstet Gynaecol 1977;84:743. 83. Turnbull AC, Anderson ABM, Flint APF, Jeremy JY, Keirse MJNC, Mitchell MD. Human parturition. In: Knight J, O'Connor M, eds. The fetus and birth. Amsterdam: Elsevier-Excerpta Medica, 1977:427. 84. Samuelsson B. Biosynthesis and metabolism of prostaglandins. In: Les Prostaglandines. Paris: INSERM, 1973:21. 85. Mitchell MD. Prostaglandins during pregnancy and the perinatal period. J Reprod Fertil 1981 ;62:305. 86. Keirse MJNC, Thiery M, Parewijck W, Mitchell MD. Chronic stimulation of uterine prostaglandin synthesis during cervical ripening before the onset of labor. Prostaglandins 1983;25:671. 87. Okazaki T, Casey ML, Okita JR, MacDonald PC, Johnston JM. Initiation of human parturition. XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua. AMJ 0BSTET GYNECO!. 1981; 139:373. 88. Okazaki T, Okita JR, MacDonald PC, Johnston JM. Initiation of human parturition. X. Substrate specificity of phospholipase A 2 in human fetal membranes. A~l J OBSTET GYNECOL 1978;130:432. 89. Bleasdale JE, Johnston JM. Prostaglandins and human parturition: regulation of arachidonic acid mobilization. Rev Perinatal Med 1984;5:151. 90. Okazaki T, Sagawa N, Okita JR, Bleasdale JE, MacDonald PC,JohnstonJM. Diacylglycerol metabolism and arachidonate release in human fetal membranes and decidua vera. J Bioi Chern 1981;256:7316. 91. Anderson ABM, Webb R, Turnbull AC. Oestrogens and parturition. J Endocrinol 1981 ;89: I 03P. 92. Ham EA, Cirillio VJ, Zanetti ME, Kuehl FA. Estrogendirected synthesis of specific prostaglandins in uterus. Proc Nat! Acad Sci USA 1975;72:1420. 93. Kuehl FA, Zanetti ME, Cirillo VJ, Ham EA. Estrogeninduced alterations in cyclic nucleotide and prostaglandin levels in target tissue. J Steroid Biochem 1975;6: 1099. 94. Chang W-C, Nakao], Orimo H, Murota S-I. Stimulation of prostaglandin cyclooxygenase and prostacyclin synthetase activities by estradiol in rat aortic smooth muscle cells. Biochim Biophys Acta 1980;620:472. 95. Fuchs A-R, Husslein P, Fuchs F. Oxytocin and the initiation of human parturition. II. Stimulation of prostaglandin production in human decidua by oxytocin. A~ J 0BSTET GYl\:ECO!. 1981;141:694. 96. Strickland DM, Kramer DL, Mitchell MD. Production of prostaglandins by human intrauterine tissues; influences of oxytocin. IRCS Med Sci 1982;10:102. 97. Bejar R, Curbelo V, Davis C, Gluck L. Premature labor. II. Bacterial sources of phospholipase. Obstet Gynecol 1981;57:479. 98. Aiken JW. Aspirin and indomethacin prolong parturition in rats: evidence that prostaglandins contribute to expulsion of fetus. Nature 1972;240:21. 99. Novy MJ, Cook MJ, Manaugh L. Indomethacin block of normal onset of parturition in primates. AM J 0BSTET GYNECOL 1974; 118:412. 100. Collins E, Turner G. Maternal effects of regular salicylate ingestion in pregnancy. Lancet 1975;2:335. 101. Lewis RB, Schulman JD. Influence of acetylsalicylic acid, an inhibitor of prostaglandin synthesis, on the duration of human gestation and labour. Lancet 1973;2: 1159. 102. Waltman R, Tricomi V, Palav AB. Mid-trimester hyper-

November 1987 Am J Obstet Gynecol

103. 104. 105. 106. 107. 108. 109. 110. Ill. 112.

113.

114.

115. 116. 117.

118. 119. 120.

121. 122. 123. 124.

125.

tonic saline-induced abortion: Effect of indomethacin on induction/abortion time. AM J 0BSTET GYNECOL 1972; 114:829. Wiqvist N, Lundstrom V, Green K. Premature labor and indomethacin. Prostaglandins 1975; I 0;515. Nieby!JR, Blake DA, White RD, eta!. The inhibition of premature labor with indomethacin. AM J OBSTET GvNECOL 1980;136:1014. Niebyl JR, Witter FR. Neonatal outcome after indomethacin treatment for preterm labor. AMJ 0BSTET GvNECOL 1986; 155:747. Stuart MJ, Gross SJ, Elrad H, Graeber JE. Effects of acetylsalicylic-acid ingestion on maternal and neonatal hemostasis. N Eng! J Med 1982;307:909. Southern EM, Gutknecht, GD, Mohberg NR, Edelman DA. Vaginal prostaglandin E 2 in the management of fetal intrauterine death. Br J Obstet Gynaecol 1978;85:437. Thiery M, Parewijck W, Decoste J-M. Prostaglandins for the management of anencephalic pregnancy. Prostaglandins 1981;21:207. Chan WY. Uterine and placental prostaglandins and their modulation of oxytocin sensitivity and contractility in the parturient uterus. Bioi Reprod 1983;29:680. Liggins GC. Hormonal interactions in the mechanism of parturition. Mem Soc Endocrinol 1973;20: 119. Whalley ET. The action of bradykinin and oxytocin on the isolated whole uterus and myometrium of the rat in oestrous. Br J Pharmacal 1978;64:21. Brummer HC. Further studies on the interaction between prostaglandins and syntocinon on the isolated pregnant human myometrium. J Obstet Gynaecol Br Comm 1972;79:526. Garrioch DB. The effect of indomethacin on spontaneous activity in the isolated human myometrium and on the response to oxytocin and prostaglandin. Br J Obstet Gynaecol 1978;85:47. Dubin NH, Ghodaonkar RB, King TM. Role of prostaglandin production in spontaneous and oxytocininduced uterine contractile activity in in vitro pregnant rat uteri. Endocrinology 1979;105:47. VaneJR, Williams KI. The contribution of prostaglandin production to contractions of the isolated uterus of the rat. Br J Pharmacal 1973;49:629. Malofiejew M, Blaszkiewicz Z. Influence of R-805 on the contractility and reactivity of rat myometrium. Arch Int Pharmacodyn 1979;238:233. Giannopoulos G, Jackson K, Kredentser J, Tulchinsky D. Prostaglandin E and F2" receptors in human myometrium during the menstrual cycle and in pregnancy and labor. AMj 0BSTET GYNECOL 1985;153:904. Carsten ME, Miller JD. Prostaglandin E 2 receptor in the myometrium: distribution in subcellular fractions. Arch Biochem Biophys 1981;212:700. Kimball FA, Kirton KT, Spilman CH, Wyngarden LJ. Prostaglandin E 1 specific binding in human myometrium. Bioi Rep rod 1975; 13:482. Hofmann GE, Rao Ch V, Barrows GH, Sanfilippo JS. Topography of human uterine prostaglandin E and F 2" receptors and their profiles during pathological states. J Clin Endocrinol Metab 1983;57:360. Garfield RE, Rabideau S, Challis JRG, Daniel EE. Hormonal control of GAP junction formation in sheep myometrium during parturition. Bioi Reprod 1979;21:999. Garfield RE, Kannan MS, Daniel EE. Gap junction formation in myometrium: control by estrogens, progesterone, and prostaglandins. AmJ Physiol1980;238:C8l. Garfield RE, Hayashi RH. Appearance of gap junctions in the myometrium of women during labor. AM J OBSTET GYNECOL 1981; 140:254. Sutherland EW, Rail TW. The relation of adenosine3',5'-phosphate and phosphorylase to the actions of catecholamines and other hormones. Pharmacol Rev 1960; 12:265. Yang JC, Triner L, Vulliemoz Y, Verosky M, Ngai SH.

Uterine muscle contraction

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126.

127. 128.

129. 130.

131. 132. 133.

134.

135.

136.

137. 138.

139. 140. 141. 142.

Effects of halothane on the cyclic 3',5' -adenosine mono-· phosphate (cyclic AMP) system in rat uterine muscle. Anesthesiology 1973;38:244. Goldberg ND, Haddo}$: MK, Hartle DK, Hadden JW. The biological role of cyclic 3',5'-guanosine monophosphate. Proc Fifth Inti Congr Pharmacal Basel:Karger, 1973;5:146. Marshall JM, Fain JN. Effects of forskolin and isoproterenol on cyclic AMP and tension in the myometrium. Eur J Pharmacal 1985;107:25. Smith DD, MarshaliJM. Forskolin effects on longitudinal myometrial strips from the pregnant rat: relationship with membrane potential and cyclic AMP. Eur J Pharmacoll986;122:29. Meisheri KD, McNeill JH, Marshall JM. Effect of isoproterenol on the isolated pregnant rat myometrium. Eur J Pharmacal 1979;60: 1. Diamond J, Holmes TG. Effects of potassium chloride and smooth muscle relaxants on tension and cyclic nucleotide levels in rat myometrium. Can J Physiol Pharmacoll975;53:1099. Meisheri KD, Tenner TE, McNeill JH. Effect of extracellular Na on isoproterenol-induced cAMP levels in depolarized uterus. Eur J Pharmacal 1978;53:9. Krall JF, Barrett JD, Korenman SG. Coupling of 13-adrenoreceptors in rat uterine smooth muscle. Bioi Reprod 1981 ;24:859. Vesin M-F, Harbon S. The effects of epinephrine, prostaglandins, and their antagonists on adenosine cyclic 3',5'-monophosphate concentrations and motility of the rat uterus. Malec Pharmacal 1974; 10:457. Carsten ME, Miller JD. Regulation of myometrial contractions. In: MacDonald, PC, Porter J, eds. Initiation of parturition: prevention of prematurity. Report of the Fourth Ross Conference on Obstetric Research. Columbus, Ohio: Ross Laboratories, 1983:166. KraliJF, SwensenJL, Korenman SG. Hormonal control of uterine contraction. Characterization of cyclic AMPdependent membrane properties in the myometrium. Biochim Biophys Acta 1976;448:578. Nishikori K, Takenawa T, Maeno H. Stimulation of microsomal calcium uptake and protein phosphorylation by adenosine cyclic 3',5'-monophosphate in rat uterus. Malec Pharmacoll977;13:671. Diamond J, Hartle DK. Cyclic nucleotide levels during carbachol-induced smooth muscle contractions. J Cyclic Nucleotide Protein Phosphor Res 1976;2: 179. Diamond]. Lack of correlation between cyclic GMP elevation and relaxation of nonvascular smooth muscle by nitroglycerin, nitroprusside, hydroxylamine and sodium azide. J Pharmacal Exp Therap 1983;225:422. Berridge MJ. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 1984;220:345. Hokin LE. Receptors and phosphoinositide-generated second messengers. Ann Rev Biochem 1985;54:205. Marc S, Leiber D, Harbon S. Carbachol and oxytocin stimulate the generation of inositol phosphates in the guinea pig myometrium. FEBS Lett 1986;201:9. Carsten ME, Miller JD. Ca 2 + release by inositol trisphosphate from Ca 2 + -transporting microsomes derived

143.

144.

145. 146.

H 7. 148.

149. 150. 151.

152.

153. 154.

155.

156.

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from uterine sarcoplasmic reticulum. Biochem Biophys Res Commun 1985;130:1027. Hashimoto T, Hirata M, Itoh T, Kanmura Y, Kuriyama H. Inositol 1,4,5-trisphosphate activates pharmacomechanical coupling in smooth muscle of the rabbit mesenteric artery. J Physiol 1986;370:605. Yousufzai SYK, Abdei-Latif AA. The effects of alpha 1 adrenergic and muscarinic cholinergic stimulation of prostaglandin release by rabbit iris. Prostaglandins 1984; 28:399. Ross EM, Gilman AG. Biochemical properties of hormone-sensitive adenylate cyclase. Ann Rev Biochem 1980;49:533. Spiegel AM, Gierschik P, Levine MA, Downs RW. Clinical implications of guanine nucleotide-binding proteins as receptor-effector couplers. N Eng! J Med 1985; 312:26. Gilman AG. Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase. J Clin Invest .' 1984;73:1. Cassel D, Selinger Z. Mechani~rp of adenylate cyclase activation by cholera toxin: i'lh!pi~io!l of GTP hydrolysis at the regulatory site. Proc N~hl .IWad Sci USA 1977; 74:3307. KraliJF, Leshon SC, Frolich M,Jamgotchian N, Karenman SG. Adenylate cyclase activation. J Bioi Chern 1982;257:10582. Cockcroft S, Gomperts BD. Role of guanine nucleotide binding protein in the activation of polyphosphoinoJitide phosphodiesterase. Nature 1985;314:534. Uhing RJ, Jiang H, Prpic V, Exton JH. Regulation of a liver plasma membrane pqosphoinositide phosphodiesterase by guanine nudeotides and calcium. FEBS Lett 1985;188:317. Sasaguri T, Hirata M, Kuriyama H. Dependence on Ca 2 + of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery. BiochemJ 1985;231:497. Dawson AP. GTP enhances inositol trisphosphatestimulated Ca 2 + release from rat liver microsomes. FEBS Lett 1985;185:147. Smith JB, Smith L, Higgins BL. Temperature and nucleotide dependence of Ca release by myo-inositol 1,4,5trisphosphate in cultured vascular smooth muscle cells. J Bioi Chern 1985;260:14413. Gill DL, Ueda T, Chueh S-H, Noel MW. Ca 2 + release from endoplasmic reticulum is mediated by a guanine nucleotide regulatory mechanism. Nature 1986; 320:461. Ueda T, Chueh S-H, Noel MW, Gill DL. Influence of inositol 1,4,5-trisphosphate and guanine nucleo~ides on intracellular calcium release within the NlE-115 neuronal cell line. J Bioi Chern 1986;261:3184. Chueh S-H, Gill DL. Inositol 1,4,5-trisphosphate and guanine nucleotides activate calcium release from endoplasmic reticulum via distinct mechanisms. J Bioi Chern 1986;261:13883.