Role of gap junctions and nitric oxide in control of myometrial contractility

Role of Gap Junctions and Nitric Oxide in Control of Myometrial Contractility Robert E. Garfield, Mariam Ali, Chandrasekhar YaUampalli, and Hidetaka Izumi

T here are many mechanisms involved in the regulation of uterine contractility. The con-

version of myometrial contractility during pregnancy f rom quiescence to an active and reactive state during labor, a condition dominated by rhythmic, synchronous and forceful contractility, is the result of a complex interaction of an array of systems and events. Normal labor, as well as abnormal, is thought to be preceded by a series of hormonal changes including deviations in levels of steroid hormones, prostaglandins, and cy- tokines. These episodes activate a number o f processes in the muscle cells that t ransform it to a higher level o f excitability and thereby modify it for contractions during labor. At the same time, there are decreases in mechanisms that depress contractility. In this article, we will briefly outline two systems thought to,be of great importance in regulating uterine contractility during pregnancy. One system consists of cell-to-cell conduction pathways termed "gap junct ions" which, when present, increase excitation of the myometrium. Many studies have shown that the formation and presence of gap junct ions between myometrial cells are necessary for labor, and the absence or closure of these specialized contacts favors inactivity. The other system is newly described and composed of an L-arginine-nitric oxide-cGMP pathway that suppresses contractility during pregnancy, but not during labor.

Gap Junctions

Studies in rats, published in the late 1970s, showed that gap junctions were low or absent throughout most of pregnancy, but that the junctions formed during term and pre term labor.l'~ Subsequent examination of o ther species, including humans, showed similar results, s'6 These observations indicated that the maintenance of pregnancy by the quiescent uterus might be achieved by the low frequency of cell-to-cell communication. In contrast, the onset and pro-

gression of labor might be accomplished by a heightened level of excitation when the junctions appeared. Other studies demonstrated that electrical and metabolic coupling were elevated when gap junctions were present during labor 7~~ and that hormones regulated the appearance of the junct ions between the muscle cells and also con- trolled the permeability of the gap junctions, s'a'l 1-14 In this short outline, we will not attempt to review this entire field, but only describe more recent developments in this area and emphasize why myometrial gap junct ions are important.

The contraction relaxation sequence of the myometr ium is a direct consequence of the cyclic depolarization and repolarization of the membranes of the muscle cells. Contractions of the muscle cells are initiated by a rise in the intracellular concentrat ion of free calcium (Ca ~+) (see first and second articles in this issue). The source of this activator Ca ~+ is extraceUular (Ca ~+ which flows into the cell down its electrochemical gra- dient in response to a change in permeability) or intracellular (Ca ~+ released from internal storage sites), or a combination of both. ~5t~ In the myometrium, the inward sodium (Na+) and Ca 2+ current during action potential depolarization opens voltage-dependent calcium channels to allow Ca 2+ to enter. Depolarization may also cause the release of Ca ~+ from intracellular storage sites and many agents release stored Ca2+. ~s Con- versely, a reduction in intraceUular free Ca 2§ a result of Ca 2+ pumping either outwardly acrossed the plasma membrane or into internal storage sites, terminates a contraction.

From the Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Gal- veston, TX. Address reprint requests to Robert E. Garfield, PhD, Director of Reproductive Sciences Division, Department of Obstetrics and Gy- necology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-1062. Copyright �9 1995 by W.B. Saunders Company 0146-0005/95/1901-0005505.00/0

Seminars in Perinatology, Vol 19, No 1 (February), 1995: pp 41-51 41

4 2 Garfield et al

Contractions of the myometrium are directly related to the underlying electrical activity described previously. The frequency, duration, and magnitude of uterine contractions are recognized as being respectively dependent on the frequency of action potential discharge, the duration of the train of action potentials within each muscle cell, and the total number of cells simultaneously and synchronously active. 19 Therefore, the propagation of potentials from pacemaker regions or from areas influenced by stimulatory agents to adjacent and distant cells is of fundamental importance in events controlling excitability and contractility.

Propagation of action potentials from cell-to- cell is essential for coordinating contractile activity of the myometrium, which is composed of billions o f small muscle cells. Gap junctions are sites of propagation or conduction of action potentials between cells of almost all tissues.2~ Gap junctions are intracellular channels that link cells to their neighbors and allow current carrying in- organic ions and small molecules to pass between cells, thereby facilitating electrical and metabolic coupling. The pores of the gap junction channels, which connect the interiors of two cells, are composed of proteins, termed "connexins." 22 Each gap junct ion can be made up from a few to thou- sands o f channels, and each channel is con- structed of six connexin proteins in one cell aligned symmetrically with six connexins in the adjacent connected cell. The gap junction proteins have been cloned, and antibodies have been prepared to the connexins. In the myometrium a 43-kd protein, termed "connexin 43" (Cx43), is thought to be the major component of the gap junction. Cx43 is also found in other tissues, including the heart where it is thought to be required for synchronization of cardiac contractility. 22,23

The onset and progression of labor contractile activity during term or preterm labor in all species studied is associated with the presence of large numbers of gap junctions between the myometrial cells. 1'2'1~ Moreover, improved electrical 9"24-26 and metabolic 7 communication between uterine smooth muscle cells is associated with the presence of junctions, supporting the hypothesis that the gap junctions permit the myometrium to behave as a functional syncytium during parturition. These alterations in the ex-

tent of structural and functional coupling are significant; therefore, the presence of gap junctions and cell-to-cell communication probably represents the biophysical basis for synchronous and effective uterine contractile activity during labor.

Many studies suggest the presence of specific physiological mechanisms for regulating and producing alterations in structural and functional coupling in the myometrium during pregnancy and parturition. We have previously described the possible mechanisms involved in the control of coupling in uterine smooth muscle: (1) the presence of gap junctions and hence the extent of structural coupling; (2) the permeability of the gap junctions, and hence the extent of functional coupling in the myometrium, and (3) the degradation of the gap junctions. 1~ The integrated function of these control mechanisms presumably operates to ensure the absence and /or closure of the junctions during most of pregnancy to maintain uterine quiescence, while the presence of the junctions during labor coordinates contractile activity in the myometrium and provides for effective delivery of the fetus(es).

Previously we applied electron microscopy, and electrophysiological and diffusion techniques to analyze myometrial tissues for the presence of gap junctions. More recently, antibodies and molecular probes have been used to assess the occurrence of myometrial gap junctions. 2731 The limit of resolution with immunohistochemistry approaches that of the electron microscope, par- ticularly if the fluorescent secondary antibody techniques are selected and methods for ampli- fication of the signal are employed. We have used Cx43 antibodies for immun0cytochemical (Fig 1) and immunoblot (Fig 2) studies of myornetrial gap junctions and probes for polymerase chain reaction (PCR) (Fig 3) for Cx43 mRNA to con- firm the studies done with electron microscopy and electrophysiology. Unfortunately, these methods do not differentiate between cell types (ie, smooth muscle vs fibroblasts) or various membranes (internal vs plasma membrane). Also, none of the methods using molecular techniques adequately defines by itself the presence and extent of functional cell-cell coupling.

Because of the small size of myometrial cells and the heterogeneity of the uterus, the degree of difficulty of most of the analytical techniques

Myometrial Gap Junctions and Nitric Oxide 43

Figure 1. Myometrial gapjuncuons in the guinea pig uterus demonstrated with connexin43 antibody during delivery. Shown is a fluorescent micrograph taken of uterine muscle. Each bright fluorescent spot represents a gap junction as an aggregation of connexin 43 proteins. Original magnification • 1000. (Reprinted with permission. ~9)

for measurement of gap junct ions in the myomet r ium and the pitfalls in interpretat ion of the results cannot be overemphasized. The most valid techniques must be those that directly assess the presence o f gap junct ions and their coupling. Electron microscopy still remains the method of choice for the measurement of gap junctions,

because this method can detect small gap junctions and allows discrimination between cell types and membranes . However, because there is no procedure for direct functional evaluation of gap junct ions af ter such identification, and because the presence of gap junct ions alone does not dis- close the extent o f electrical or metabolic coupling, a combinat ion of these methods is pre- ferred.

The state of phosphorylat ion of connexin has been correlated with changes in functional states of gap junct ion communicat ion in some cases, s~ Studies of several tissues suggest that there are multiple sites for phosphorylat ion. 21'~2"s4 Phos- phorylation of some types of connexins is thought to lead to the open configurat ion in some cells, whereas the dephosphoryla ted fo rm of the same connexin is associated with the open state in other cells. O the r studies indicate that cAMP-dependent protein kinases may regulate phosphorylation. 32 At present, there is no evidence for such a mechanism by myometrial cells, but it is likely that the effects o f cAMP to close the junct ions 8'~n are mediated in such a manner . I t has also been suggested that connexin phosphorylation may be involved in the assembly of gap junctions, s4's6 Western blots o f Cx43 in myometrial tissues show characteristic banding pat terns typical o f phosphorylated and unphosphoryla ted forms of the

Figure 2. Cx43 protein abundance during gestation. (A) Immunobiot of rat myometrial microsomal frac- tions from various times of gestation using a Cx43 antibody and en- hanced chemiluminescence (ECL) detection system. Abbreviations: NP, nonpregnant; d16, d19, day of pregnancy in which d22 is term; Dei, delivering; PPI and PP3, postpartum days as compared with heart and liver; UP, unphosphorylated band; PI and P2, phosphorylated bands. (B) Bar graphs of densio- metric estimates of immunoblot at various times of gestation showing absolute densities of each of the three bands. (From Byam-Smith, Ali and Garfield, unpublished.)

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Figure 3. Rat myometrial Cx43 mRNA abundance during gestation. (A) Photograph of PCR products run on 8% acrylamide gel and are stained with ethidium bromide. Rat myometrial RNA from different days of gestation. Abbreviations: 22NL, term non-labor; 22L, term laboring; P1 and P2, postpartum 1 and 2 days; NRT, no reverse transcriptase control; NR, no RNA control; Cont, kit control; M, markers. PCR conditions were 22 cycles of denaturation at 94~ for 15 seconds; annealing at 57~ for 30 seconds; and at 72~ for 45 seconds. Cx43 product is 140 base pairs and internal standard is 630 base pairs. (B) Bar graphs show relative abundance of Cx43 mRNA during gestation. Photographs were densiometrically scanned, giving density values. Each Cx43 value was normalized to the internal standard value within each lane. (From Ali and Garfield, unpublished.)

protein (see Fig 2). The mechanisms regulating phosphorylat ion and conditions favoring phosphorylation need fur ther study.

Although it has been shown that gap junct ion format ion in myomet r ium is under hormonal control such as stimulation by estrogen and inhibition by progesterone, 1'2'1]'1s'~4 the precise mechanism of gene regulation by these agents is currently not clear. Fishman et al ~3'37 and Miller et al 3s showed that Cx43 and Cx32 have a similar genomic s t ructure and are composed of two ex- ons: the first exon is small and is located in the 5' untranslated sequence; the second exon contains the entire coding sequence. It has been suggested by many 22'a9 that there is no interrup- tion in the coding sequence by introns. Cx43 has been mapped to chromosome 6, 4~ and its pseu- dogene is m a p p e d to chromosome 5. 41

Recent studies have also a t t empted to define the cis-acting elements of the Cx43 gene that regulate its synthesis. F rom the human Cx43 gene, a series o f chimeric luciferase repor te r genes containing nested deletions were con- structed. 42 This study showed that p romo te r constructs were active when propaga ted in vitro and suggested that the proximal p romote r may confer tissue specifity. Additionally, the p romoter region of the Cx43 gene f rom rats has been cloned recently. 4a The results o f this study indicate that the p romo te r region contains a TATA box, AP-1 and AP-2 sites (sequences thought to mediate effects o f cAMP), and half-palindromic

estrogen response elements. When the repor te r was linked to a r epor te r gene luciferase, it increased the expression of Cx43 in H e L a cells transfected with the luciferase-Cx43 construct. When H e L a cells were cotransfected with estrogen receptor cDNA, an upregulation of luciferase expression occurred when estrogen was present. This study demonstra tes that estrogen response elements are present on the 5' region of p romoter region of the Cx43 gene, and suggests that es- t rogen may regulate expression o f the gene pro- vided estrogen receptors are present. This ob- servation may account for the ability o f estrogen to stimulate gap junctions in the myometrium 1H4 and raise Cx43 mRNA levels. 31 However, clearly estrogen is not required for this process because gap junct ions will develop in the myomet r ium in vitro in the complete absence of estrogen. 12

Cx43 cDNA clones have been made for heart and uterine t i s sues . 22'44'45 Clones for Cx43 have been used by several groups to study expression of gap junct ion transcripts in the myomet r ium during pregnancy. 22'45 Generally, there are con- flicting data regarding the presence of gap junctions, Cx43 levels, and the change in expression of Cx43 mRNA in the rat uterus during pregnancy. Cx43 mRNA levels change little during pregnancy despite a 50-fold to 100-fold increase in myometrial gap junct ions dur ing labor as ev- idenced by electron microscopy and immuno- cytochemistry of Cx43. Risek et al ~s and Lye et al sl measured about a fourfold to fivefold in-


crease in Cx43 mRNA at term in the uterus, whereas Lang et al 4~ concluded there was little or no increase in levels of Cx43 mRNA during delivery. Similarly, we have found only a small increase in Cx43 mRNA during delivery compared with predelivery values (Fig 3).

There are obvious reasons for some of the above differences, such as the following: (1) not all groups have taken uterine tissues at the same times or even during delivery; (2) not all analyses were normalized in the same fashion; (3) in some studies, whole uterine tissues were examined and not simply the myometrium; and (4) some studies used crude homogenates whereas others used more purified membranes. However, all studies indicated little change in mRNA levels before or during labor. In our studies, only myometr ium was used and Cx43 mRNA was normalized to an internal Cx43 standard. Thus, our results and those of Lang et al 4"~ showed that mRNA levels for Cx43 increase little before or during labor despite a t remendous increase in muscle gap junct ion levels and about a 15-fold increase in Cx43 protein levels (Fig 2).

The above information suggests that regulation of Cx43 proteins into gap junct ions is not predominantly under transcriptional control. Although for most genes transcriptional control is the most important pathway, cells can control the proteins they make by other mechanisms, including the following: (1) regulation of how the primary RNA transcript is spliced or otherwise processed (RNA processing control); (2) selection o f mRNAs that are transported to the nucleus (RNA transport control); (3) instability o f certain mRNA molecules in the cytoplasm (mRNA sta- bility control); (4) selection of mRNAs translated by ribosomes (translational control); and (5) activation, inactivation, or sequestration of specific protein molecules after they have been made (protein activity control). 46 It is possible that any one of these steps may be involved in the regulation of Cx43 proteins and gap junct ions in myometrial cells during labor.

Gap junct ions play important roles as sites for the coordination of action of substances which either stimulate or inhibit the myometr ium to contract. Agents that increase or decrease myometrial contractility can influence the muscle cells in a number of different ways that may or may not directly affect the gap junctions. However,

these agents do not work independently of the gap junct ions and the propagation of action potentials. As noted previously, the influx and efflux of Ca 2+ ions in the muscle cells during the passage of the action potentials, and the opening of voltage-dependent Ca ~+ channels, are responsible for the cyclical increases and decreases in tension. This mechanism can be modified by endogenous or exogenous hormones, neurotransmitters , and other agonists and antagonists to ei ther increase or decrease the excitability o f the muscle and thereby raise or lower the ability o f action potentials to propagate. Thus, the effects of excit- atory and inhibitory agents are superimposed upon the driving force that is supplied by the propagation of action potentials. Gap junct ions are also involved in regulation of relaxation (inactivity) of the myometrium. As mentioned previously, the propagation of electrical signals is key to control of contractility. I f propagation or conduct ion is low, contractility will be severely depressed. In addition, many inhibitory substances can indirectly affect these systems in a number of ways, but like contractile agonists they do not function independently of gap junctions and the propagation of action potentials. Lack o f conducted action potentials and inactivity of the myometr ium are promoted by decreases in (1) pacemaker activity, (2) excitability of the muscle cells, and (3) cell-to-cell coupling. Any agent that suppresses the generation of action potentials, closes gap junctions, or decreases their number inhibits the active events and thereby af- fects contractility. These contractile antagonists may act to prevent activity not by causing relaxation of the myometrium, but by prevention of contractile activity o f the muscle.

We have briefly reviewed the evidence that gap junct ions play an essential role in the gradual evolution of uterine contractility during labor and the significance of increased intercellular coupling. Our studies and others suggest that the synthesis, degradation, and permeability of myometrial junctions are physiologically regulated by hormones and that the junctions can be regulated pharmacologically to control term or pre term labor.

Inhibition of Myometrial Contractility by Nitric Oxide The discovery that nitric oxide, a short-lived free radical gas, is involved in controlling many bio-

46 Garfield et al

logical function including relaxation of various smooth muscles such a s v a s c u l a r , 4749 intestinal, ~~ tracheal, 51 and corpus cavernosa152 is one of the most exciting and significant recent advances in biology and medicine. However, until presently there were no studies that indicated that nitric oxide might regulate uterine contractility, except that nitroglycerin and sodium nitroprusside have been shown to inhibit contractions 5a and it is now recognized that these two compounds are nitric oxide donors.

We recently examined the possibility that nitric oxide might be one of the factors that mediate uterine relaxation dur ing pregnancyP 4~6 We tested the effects o f L-arginine, the substrate for nitric oxide, on uterine contractility of strips of tissues f rom pregnant rats in vitro. L-arginine (Fig 4A) and nitric acid (Fig 4B) caused a rapid and substantial relaxation of spontaneous activity of the uterine strips f rom rats at mid to near t e rm gestation (Fig 4). The relaxation effects were re- versed by L-nitro-arginine methyl ester (L- NAME), an inhibitor o f nitric oxide synthase. Sodium nitroprusside, a nitric oxide donor , completely abolished spontaneous contraction. Methylene blue, an inhibitor of guanylate cyclase, also prevented the inhibitory effects of L-arginine. These results strongly support the existence of an L-arginine-nitric ox ide-cGMP system for regulating uter ine relaxation. At the same time these studies were published, several other papers confirmed the existence of this pathway in the u t e r u s . 57-59

We also examined the effects of L-arginine on tissues f rom delivering animals and were sur- prised to find that L-arginine had little effect. Figure 5A shows dose responses (dose of L-arginine vs durat ion o f inhibition) of L-arginine of tissues at day 18 to day 22 of pregnancy, dur ing delivery and postpartum. These studies show that the inhibitory action of the L-arginine was con- siderably lower dur ing delivery and may indicate that nitric oxide may contr ibute to the maintenance of uterine quiescence during pregnancy but not dur ing delivery.

Because responses to L-arginine were lower at delivery than pre term, we reasoned that perhaps the generat ion o f nitric oxide by nitric oxide synthase is lower dur ing delivery, that guanylate cyclase produces less cGMP during delivery, or that cGMP is less effective during delivery. When we

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tested the ability of 8-bromo-cGMP to inhibit uter ine contractions, we found that dur ing delivery at te rm or pre term, the responses were greatly at tenuated versus p re t e rm nondelivery (Fig 5B). These data clearly indicate that the effector system of cGMP to produce relaxation is greatly reduced dur ing te rm and p re t e rm delivery. The fact that the ant iprogesterone com- pound (ZK299, Schering, Berlin, Germany) produced similar changes to those at term shows that the decrease in sensitivity to cGMP may be con- trolled by progesterone (Fig 5B). This finding has b roade r implications in that o ther substances which inhibit uterine contractility may act through cGMP and be expected to decline during labor.

The exact cellular mechanisms of the cGMP- dependent relaxation system in myometrial tissues is unknown. We have evaluated the effects o f cGMP on myometrial contractility in very small strips of myometrial tissues. 54 A permeable an-


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Figure 5. Effects of (A) L-arginine and (B) 8-bromo-cGMP on uterine contractions in vitro. (A) Dose-dependent relaxation effects of L-arginine (0.1 mM to 10 mM) on spontaneously contracting uterine strips from rats at days 17 to 21 of gestation and on day 22 with and without delivery (d22 del) and postpartum days 1 and 2 (dl pp and d2 pp). The duration of inhibition of spontaneous uterine contractions are dose-dependent. The effects of 1.-arginine from concentrations of 1 mM are significantly (P < .01) decreased during spontaneous delivery compared with days 16 to 21. Each data point represents mean +SEM. The total number of strips studied at each time period was 8 to 16 from four to six animals per group. (Reprinted with permission. 56) (B) Effect of 8- bromo-cGMP on contraction of uterine strips in vitro from rats at day 19 of pregnancy (closed circles), day 18 of pregnancy during preterm birth following antiprogesterone treatment (ZK299, Schering) (open squares), and during spontaneous birth at term (open circles). Note that during both term and preterm labor, the dose- response relationship to cGMP is shifted to a less sensitive state. (Reprinted with permission. 5~)

alog o f cGMP (8 b romo-cGMP) inhibi ted carbachol-induced contract ions but not KCI- provoked activity (Fig 6). In addition, 8-bromo- cGMP and sodium nitroprusside reduced carbachol-evoked contractions in Ca ~+ free solutions (Fig 7). The effects o f sodium nitroprusside and 8-bromo-cGMP were decreased respectively by methylene blue (an inhibitor o f guanylate cyclase) and Rp-8-bromo-cGMPs (a competi t ive inhibitor o f cGMP). Because carbachol, but not KCI, produces contract ions of the myomet r ium by re- leasing internal Ca ~+ f rom storage sites, we pro- pose that cGMP acts primarily by interfering with Ca ~+ release f rom within the myometrial cell. However, cGMP also inhibits contractions in skinned myometrial tissues, indicating that it may have an effect directly on interaction o f the myo- filaments.

In o rder to evaluate if the generat ion levels o f nitric oxide and cGMP were elevated during pregnancy and decreased dur ing parturi t ion, we measured the product ion o f nitric oxide and cGMP by uterine tissues f rom rats ei ther nonpregnant or at different stages of gestation. Total nitrites p roduced by uterine tissues were low in nonpregnant rats (100%), substantially elevated during the mid stage of gestation (600%), and dramatically decreased at the time of spontaneous delivery (3%) and day 1 pos tpa r tum (10%)

(Fig 8). The uterine content o f cGMP in the same tissues followed a similar trend, cGMP levels were increased dur ing pregnancy and decreased during partur i t ion and pos tpar tum (Fig 8). These studies provide persuasive evidence to indicate that uter ine nitric oxide product ion and cGMP generat ion are closely linked.

To test the possibility that the nitric oxide in- hibitors or donors might ei ther initiate p re t e rm labor or prevent the onset o f spontaneous labor respectively, we treated intact p regnan t rats at day 17 through to te rm with L-NAME or nitroglycerin. We found that nei ther c o m p o u n d al- tered the timing of parturit ion: all animals deliv- e red spontaneously at term along with controls. The fact that nei ther nitric oxide synthase inhib- itors nor donors initiated early labor or prevented te rm labor suggests that nitric oxide may not be essential for the maintenance of pregnancy and its withdrawal may not initiate labor. However , nitric oxide may still play a significant role in uterine quiescence, and nitric oxide donors might be effective in suppressing uter ine contractility. We suggest that nitric oxide inhibits uter ine contractility before conversion o f the myomet r ium to an active and reactive muscle to p roduce effective labor contractions. Labor is achieved when the inactive muscle is activated by changes in steroid hormones and the addition of gap

4 8 Garfield et al

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Figure 6. Effects of various concentrations of 8- bromo-cGMP on 45 mM KCl-induced contractions (Aa) and on 1 #M carbachol contractions (Ab). KC1 and carbachol were applied to myometrial strips for 1 minute at 10-minute intervals. Various concentrations (1 pM to 0.1 mM) of 8-bromo-cGMP were applied cu- mulatively for 10 minutes. (Aa) Effects of 8-hromo- cGMP on KCl-induced contractions (Ab) and on carbachol contractions. (B) Relative tensions produced by KC1 (triangles) or carbachol (circles) with 8-bromo- cGMP at three concentrations. The amplitudes of KC1- or carbachol-induced contractions before application of 8-bromo-cGMP were used as 1.0 to normalize other values. Values represent the means; vertical bars indicate SE; n = 6 to 7 animals. (From Izumi and Gar- field, unpublished.)

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Figure 7. Effects of sodium nitroprusside (SNP) and 8-bromo-cGMP on 1 ~tM carbachol-induced contractions in Ca 2+ free (2 mM EGTA-containing) solutions. Carbachol was applied to myometrial strips in Ca 2+- free solutions to deplete the Ca 2+ in storage sites. The solution was changed to Krebs' (Ca2+-containing) solution for 20 minutes and then the solution was again changed to Ca2+-free solution. Carbachol was added 1 minute after again changing to Ca2+-free solution (Aa and Ba). (Ab) and (Bb) demonstrate the action of 1 mM of SNP for 20 minutes (Ab) and 0.1 mM 8- bromo-cGMP for 10 minutes (Bb) when applied to the same strips shown in (Aa) and (Ab). (C) shows the effects of various concentrations of SNP either alone (A), with 10 #M methylene blue (MB) (A), or with 10/zM Rp-8- bromo-cGMPS (n), and the effects of various concentrations of 8-bromo-cGMP without (�9 or with 10 #M Rp-8-bromo-cGMPS (O) on the carbachol-induced contractions in Ca2+-free solutions. MB alone or with Rp-8-bromo-cGMPS was added 30 minutes before the application of carbachol. The amplitudes of carbachol- induced contractions before application of either SNP or 8-bromo-cGMP were used as 1.0 to normalize other contraction values. Values represent the means; vertical bars indicate SE; n = 5 to 6 animals. (From Izumi and Garfield, unpublished.)

Myometrial Gap Junctions and Nitric Oxide 4 9

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Figure 8. (A) Total nitrites (nitrites + nitrates) produced by uterine tissues from nonpregnant (NP); pregnant on day 18 of gestation (NON DEL); spontaneously delivering at term (DEL), and day 1 postpartum (PP) rats. Values are means +_ SEM for tissues from six animals in each group. Bars with different letters at the top vary significantly. (B) Tissue cGMP content of full-thickness uterine tissues obtained from nonpregnant (NP); pregnant, on day 18 of gestation (NON DEL); spontaneously delivering (DEL) at term, and day 1 postpartum (PP) rats. Values are means + SEM for tissues from 4 to 6 animals in each group. Bars with different letters at the top vary significantly. (Reprinted with permission from YallampaUi C, et al: Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinology 133:1899-1902, 1993, �9 The Endocrine Society. ss)

ven t ing the in i t ia t ion of l abor because they act at a step before convers ion to the active stage and because of a decrease in the cGMP relaxat ion system. O n the o the r hand , ni t r ic oxide synthase inhib i tors may increase s p o n t a n e o u s cont rac-

tions, bu t they may not initiate labor because they also act before the convers ion step. Nitric oxide would no t be expected to inhib i t l abor because there is a decrease in the responsiveness of the cGMP effector system at this time.

Much work remains to be d o n e on the u t e r i ne ni tr ic oxide pathway, such as ident i f ica t ion of the types of ni tr ic oxide synthases and the effects o f h o r m o n e s on various steps. I t is likely that we will l ea rn cons iderably f rom these studies, a nd p robab ly most o f this knowledge can be appl ied to o the r tissues. It seems that ni t r ic oxide plays signif icant roles in women ' s health.

References 1. Garfield RE, Sims S, Daniel EE: Gap junctions: Their

presence and necessity in myometrium during gestation. Science 198:958-960, 1977

2. Garfield RE, Sims SM, Kannan MS, et al: Possible role of gap junctions in activation of myometrium during parturition. AmJ Physiol 235:C168-179, 1978

3. Garfield RE, Rabideau S, Challis JRG, et al: Hormonal control of gap junction formation in sheep myometrium during parturition. Bioi Reprod 21:999-1007, 1979

4. Garfield RE, Hayashi RH: Appearance of gap junctions in the myometrium of women during labor. Am J Obstet Gynecol 140:254-260, 1981

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Role of gap junctions and nitric oxide in control of myometrial contractility