0026-895X/04/6505-1051–1059$20.

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MOLECULAR PHARMACOLOGY Vol. 65, No. 5
Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3026/1149361
Mol Pharmacol 65:1051–1059, 2004 Printed in U.S.A.

MINIREVIEW

Regulation of Uterine Function: a Biochemical Conundrum in

the Regulation of Smooth Muscle Relaxation

Iain L. O. Buxton
Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada

Downloaded from molpharm.aspetjournals.org at ASPET Journals on April 22, 2015

Received October 13, 2003; accepted February 18, 2004 This article is available online at http://molpharm.aspetjournals.org

ABSTRACT
Premature birth accounts for the majority of fetal morbidity and derstanding. We review the evidence that nitric oxide-mediated
mortality in the developed world and is disproportionately rep- relaxation of myometrial smooth muscle, unlike vascular or
resented in some populations, such as African Americans in the gastrointestinal smooth muscle, is independent of global ele-
United States. The costs associated with prematurity are stag- vation of cyclic guanosine 5⬘-monophosphate. Applying our
gering in both monetary and human terms. Present therapeutic current understanding of microdomain signaling and taking
approaches for the treatment of labor leading to preterm deliv- clues from genomic studies of pregnancy, we offer a framework
ery are inadequate and our understanding of the regulation of in which to view the apparent conundrum and suggest testable
myometrial smooth muscle contraction-relaxation is incom- hypotheses of uterine relaxation signaling that can explain the
plete. The ability of nitric oxide to relax smooth muscle has led mechanistic distinctions. We propose that understanding these
to an interest in employing nitric oxide-donors in the treatment mechanistic distinctions in myometrium will reveal molecular
of preterm labor. Fundamental differences exist, however, in targets that are unique and thus may be explored as therapeu-
the regulation of uterine smooth muscle relaxation and that of tic targets in the development of new uterine smooth muscle-
other smooth muscles and constitute a conundrum in our un- specific tocolytics.

The precise physiological processes leading to birth are goal of improving our understanding of the regulation of relax-
mysterious and the physiology of preterm labor (PTL) is ation. Working first in guinea pig, then monkey, and now with
unknown (Buxton et al., 2000). The majority of PTL becomes an emphasis in human tissues, we have concluded that uterine
preterm delivery (PTD), accounting for 9 to 11% of births in smooth muscle is neither vascular nor gastrointestinal smooth
the United States (ACOG Bulletin, 2003). If a test were muscle. Beyond the obvious absurdity of this statement lies a
available to predict PTL as well as its onset, we would fail to biochemical conundrum. That is, studies of receptor signal-
prevent the delivery of a preterm fetus, because there is no transduction in these other muscles does not teach us what we
safe and effective means of halting labor and maintaining need to know about myometrium; therefore, if the critical prob-
pregnancy until term. Although various tocolytics are in rou- lem of treating PTL and PTD is to be solved, we must focus on
tine use, their efficacy and safety are questionable. basic studies in myometrium, preferably human myometrium.
Thus, a basic understanding of the mechanisms regulating The following pages describe a conundrum in cyclic nucleotide
the contractile state of uterine smooth muscle will have imme- signaling that grew out of these observations of the capacity of
diate and important clinical utility. For several years now, we nitric oxide to relax myometrium.
have been focused on studies of myometrial function with the

Nitric Oxide and Uterine Function

This work was supported by grants from the Clayton Foundation for Re-
search, the Robert Z. Hawkins Foundation, and the National Institutes of Interest in the ability of NO donors to relax myometrium
Health. (Kuenzli et al., 1996, 1998; Bradley et al., 1998; Buxton et al.,

ABBREVIATIONS: PTL, preterm labor; PTD, preterm delivery; sGC, soluble guanylate cyclase; BK, large conductance potassium channel; SK,
small conductance potassium channel; PKG, protein kinase G; rMLC20, 20-kDa regulatory myosin light chain; MLCK, myosin light chain kinase;
MP, myosin phosphatase holoenzyme; MBS, myosin-binding subunit; PP1, myosin phosphatase; ROK, Rho kinase; SIN-1, 3-morpholinosyd-
nonimine; SNAP, S-nitroso N-acetyl penicillamine; PGC, particulate guanylyl cyclase; NAADP, nicotinic acid adenine dinucleotide phosphate.

1051
1052 Buxton

2001) has led some to claim its use as a tocolytic (Lees et al., tion by NO, may be involved (Modzelewska et al., 1998;
1994). Whether NO is produced in the uterus to maintain Buxton et al., 2001).
uterine quiescence is an unanswered question. Although The notion that cGMP does not subserve all of the actions
functional data in animals suggests a role for endogenous NO of NO is now accepted. In vascular smooth muscle, where NO
in regulating aspects of normal gestation and parturition and NO signaling were first worked out, it is evident that
(Tiboni et al., 2001), efforts to detect nitric-oxide synthase in some preparations exhibit significant components of the re-
human uterus using antibodies to each of the known forms of laxation to NO that are resistant to blockade of cGMP accu-
the enzyme were negative (Bartlett et al., 1999; M. E. Brad- mulation (Eckman et al., 1994). These exceptions are now
ley and I. L. O. Buxton, unpublished observations), although seen in a variety of systems, such as renal arterioles (Trottier
there is data to the contrary (Bao et al., 2002). Still others et al., 1998), cerebral microvessels and pituitary hormone
have concluded that NO is not an endogenous mediator of secretion (Pinilla et al., 1998), regulation of neuronal cell ion
human uterine quiescence (Jones and Poston, 1997). How- channels (Ahern et al., 1999; Summers et al., 1999), and
ever, high levels of nitric-oxide synthase activity have been apoptosis (Brune et al., 1996). Exceptions are also seen in
noted in the villous trophoblast during the first trimester, nonvascular smooth muscle. In canine airway, Janssen et al.
and this activity seems to decrease toward the end of gesta- (2000) have suggested that the cGMP-independent actions of
tion (Sanyal et al., 2000). The NO thus produced or theoret-
NO donors can be ascribed to the chemistry of the NO species
ically available from endothelium or some other nonmuscle
liberated. Their data, together with the work of Jones et al.

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cell compartment would be near the uterine myometrium
(1994), suggest that the actions of NO are cGMP-dependent
and might act as a paracrine agent in the maintenance of
or -independent in airway based on the NO species delivered
uterine quiescence during pregnancy. NO is also synthesized
by a particular donor. Although such a result may be unex-
by macrophages associated with the decidua during preg-
pected based on the chemistry of NO in warm, oxygenated
nancy (Vince et al., 1990). Even cervical function during
pregnancy seems to be at least partly under NO control physiological buffers (Stamler et al., 1992; Kishnani and
(Ekerhovd et al., 1998). However, a recent review of available Fung, 1996), their finding that there is a possible role for
controlled clinical studies emphasizes that although nitro- calcium release in the cGMP-independent actions of NO is
glycerin reduced the incidence of PTD, the result was not both awkward (calcium elevation ought to signal contraction)
dramatic (Duckitt and Thornton, 2002). The issues of endog- and intriguing, and it is seen in myometrium (Tichenor et al.,
enous NO synthesis and the effects of NO donors in relaxing 2003).
laboring myometrium are not the subject of this review. The principal mechanism now established for NO signaling
Rather, this review will explore the mechanism(s) of action of that is not cGMP-dependent is that of S-nitrosylation of
NO in modulating contractility of uterine smooth muscle. proteins (Davis et al., 2001; Ahern et al., 2002). S-nitrosyla-
tion occurs nonenzymatically on the thiol side chains of cys-
teine residues. Some cysteine side chains are particularly
Nitric Oxide Signaling reactive to NO as a result of accessibility at the surface of the
folded protein, the specific chemical environment and a pu-
It is no longer accurate to attribute NO-mediated relax- tative polar S-nitrosylation consensus sequence (Jia et al.,
ation in myometrium to the actions of global elevations in 1996; Lander et al., 1997; Stamler et al., 1997). Although
cGMP. Many assume that NO acts in myometrium as a S-nitrosylation might be involved in such events as the re-
relaxing agonist in the same manner it does in other smooth lease of calcium in microdomains in which potassium chan-
muscles, through soluble guanylyl cyclase (sGC) activation nels might be activated, no such direct evidence of this is
and cGMP accumulation (Yallampalli et al., 1994; Buhimschi
available for myometrium.
et al., 1995), even though numerous studies show that cGMP
Despite the growing evidence that there are actions of NO
is neither necessary nor sufficient for myometrial relaxation
other than elevation of cGMP and that these other pathways
(Diamond, 1983; Kuenzli et al., 1996, 1998; Bradley et al.,
are present in smooth muscle, the finding that little or no
1998; Hennan and Diamond, 1998, 2001; Buxton et al., 2001;
NO-mediated relaxation is caused by sGC-cGMP accumula-
Tichenor et al., 2003). A conundrum exists: why don’t global
tion in myometrium is puzzling. The notion that relaxations
elevations of cGMP signal relaxation of myometrium as they
do in vascular smooth muscle? of myometrium are entirely cGMP-independent has drawn
Data in guinea pig uterine smooth muscle demonstrated skepticism; we too, have approached with caution the hy-
that an NO donor produced relaxation despite the inhibition pothesis that cGMP is neither necessary nor sufficient to
of sGC by methylene blue (Kuenzli et al., 1996). Further- relax myometrium. Our cautions notwithstanding, the hy-
more, concentrations of permeable cGMP analogs in excess of pothesis is supported by data other than our own (Diamond,
10 ␮M were required to produce any demonstrable relaxation 1983; Word et al., 1991; Word and Cornwell, 1998; Mod-
of the uterine smooth muscle. In both monkey and human zelewska et al., 1998). Efforts recently have been centered on
myometrium, where no relaxation can be demonstrated with the notion that ion channels, those carrying the major hyper-
cGMP analogs, NO-induced relaxation is independent of a polarizing potential in myometrial smooth muscle (Fig. 1),
sGC-cGMP pathway (Kuenzli et al., 1998; Bradley et al., calcium activated potassium channels (KCa), might be re-
1998; Buxton et al., 2001). These and other studies in which sponsible for the actions of NO to relax myometrium (Maz-
sGC-independent actions of NO have been noted suggest that zone et al., 2002). This notion is supported by the finding that
intracellular cGMP elevation is neither necessary nor suffi- scorpion toxins known to block KCa, prevent the relaxation to
cient for NO-induced relaxation of the uterine smooth muscle NO (Buxton et al., 2001) and that these channels can be
and that other pathways, such as ion channel/pump regula- activated by NO in myometrium (Shimano et al., 2000).
 A Biochemical Conundrum in Smooth Muscle 1053

KCa-Channels AF395661, AY040849, AY044441, AY049734, and AF39717).

There are differences between those sequences and ones pre-
That KCa channels are at the root of the cGMP-indepen- viously published for the human slow conductance channels
dent relaxing action of NO is intriguing and is consistent (SK2 and SK3) that would be predicted to influence their
with the cGMP-independent actions of NO described thus far voltage dependence. No evidence for a previously unde-
(Ahern et al., 1999; Mazzuco et al., 2000; Yu et al., 2002). The scribed KCa channel has been found in myometrium, which
notion that myometrial KCa are regulated directly by NO is expresses each of the known Ca2⫹-activated K⫹ channel pro-
supported by evidence in vascular (Bolotina et al., 1994) and teins; the large conductance channel (BK␣, BK␤), an inter-
gastrointestinal smooth muscle cells (Lang et al., 2000) that mediate conductance channel (IK), and SK2 and SK3.
these channels can be modulated by S-nitrosylation. This More recently, the hypothesis that KCa expression may be
hypothesis is challenging however, if based solely on the use correlated with the timing of myometrial contraction during
of KCa channel inhibitors. Blockade of channels known to birth has been explored. In particular, some have proposed
carry significant hyperpolarizing current in smooth muscle that expression and/or critical electrophysiological properties
(McCarron et al., 2002) may not offer a direct assessment of of the large conductance KCa (BK or Maxi-K) channel are
the mechanism of action of NO (Kaczorowski et al., 1996). down-regulated before birth in both animals and humans
The Ca2⫹-activated K⫹ channels from human myometrium (Khan et al., 1993, 2001; Benkusky et al., 2000; Chan-
have been cloned and sequenced (GenBank accession nos. rachakul et al., 2003). Although down-regulation of BK chan-

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nels is logical based on the fact that the BK channel carries
far more hyperpolarizing current than other members of this
channel family (200 –300 pS versus 20 –30 pS), we find no
change in the expression of BK␣ or BK␤ transcripts before
labor in humans. There are, on the other hand, changes in
the expression of slow conductance channels. Although the
regulation of SK channel activity alone is unlikely to be at
the basis of the enigma presented by the actions of NO in
myometrium, it is possible that decreased SK channel ex-
pression in myometrium is part of the accommodation at
term that subserves parturition (Mazzone et al., 2002).
Contributing to the conundrum, there is evidence that
cGMP activation of PKG leads to modulation of KCa through
both phosphorylation (White, 1999; Klyachko et al., 2001)
and dephosphorylation (White et al., 1993). Because blockade
of cGMP elevation does not alter the relaxation of myome-
trium to NO-donors, we must exclude these possibilities for
myometrium unless we are to propose that cGMP elevation
and kinase activation were to occur in a compartment of the
cell near the membrane and that such changes occurred in
the immediate proximity of the channel. Such a compartment
would not be represented by global elevation of cGMP elicited
by the actions of NO.

Compartmentation of Signaling
Such a notion is not all that improbable even if it is not a
compartment that subserves the effects of NO, but might
rather serve the actions of agonists that act by activating
Fig. 1. Proposed model of NO signaling in a uterine smooth muscle cell. particulate guanylate cyclase and elevating cGMP in a dis-
The liberation of either nitric oxide radical (NO䡠) or a redox form of NO* crete signaling domain such as the myocyte caveolae or lipid-
results in the expected activation of sGC and the accumulation of cGMP. rich raft region. This notion is attractive because recent
Activation of PKG by cGMP results in the activation of the kinase and the
studies of genes that seem to turn on between the start of
subsequent phosphorylation of substrate proteins in the cell but not those
associated with relaxation. The proposed protein-protein interaction that pregnancy and term include the gene for uroguanylin, the
is thought to occur between PKG and the myosin phosphatase that leads endogenous activator of the C-type particulate guanylate
to its activation and ability to dephosphorylate the myosin regulatory cyclase. Exploring this notion will take time, but we think it
light chains (not shown) must be questioned in myometrium because
elevation of cGMP does not relax the tissue. This interaction between may explain some of the confusion regarding a role for cGMP
PKG and myosin phosphatase is known to be critical in other smooth in the tissue.
muscles because inhibition of the myosin phosphatase leads to increased If there were a compartment in the cell that signaled
force for a given concentration of calcium (calcium sensitization). The
ability of SNAP but not SIN-1 to generate NO*-mediated release of
through cGMP (albeit not global elevations secondary to the
calcium from sarcoplasmic reticulum (SR) leads to the activation of po- action of NO on soluble guanylyl cyclase), it would require a
tassium channels (KCa) and the extrusion of potassium ions leading to compartmentation of PKG as well as the cyclase and the
hyperpolarization of the cell membrane. Hyperpolarization of the mem- channel. Despite earlier assertions that PKG subserves the
brane inactivates the inward movement of calcium through the voltage-
sensitive channel (ICa) lowering the intracellular calcium concentration relaxation of myometrium to global elevation of cGMP after-
and relaxing the muscle. treatment by NO or NO donors, little is known regarding the
1054 Buxton

isotypes of PKG in myometrium. This is particularly glaring in phasic smooth muscle and are of interest in myometrium
given the role of kinase localization that explains, in part, the because the muscle must maintain tone for extended periods
compartmentation of cAMP action in cardiac myocytes between relaxations during parturition. If Zip kinase exists
(Steinberg and Brunton, 2001). in myometrium however, it must reside in a particulate re-
Because global elevations of cGMP do not explain NO- gion of the cell, because we do not find any evidence by
mediated relaxation in myometrium, it is possible that there Western blot for the presence of Zip kinase in myometrial
is some difference in the regulation of the smooth-muscle homogenates. This could be a result of the general difficulty
myosin phosphatase. Smooth muscle contraction is initiated in finding low-abundance proteins in homogenates, in that
by phosphorylation of the 20-kDa regulatory myosin light the original description of the kinase was in samples first
chain (rMLC20) by a Ca2⫹/calmodulin dependent activation isolated as myofibrillar pellets. The presence and distribu-
of the myosin light chain kinase (MLCK) which phosphory- tion of Zip kinase in myometrium will be interesting to dis-
lates rMLC20 on Ser-18,19, leading to an acceleration of cover because we propose that a nonzipper isoform of MBS is
actin-myosin ATPase (Stull et al., 1991). Smooth muscle re- present in the cell soluble fraction and thus would not be
laxation is in large measure the result of dephosphorylation expected to interact with and be phosphorylated by a Zip
of rMLC20 by myosin phosphatase holoenzyme (MP). MP is a kinase. Indeed, with appropriate controls from gastrointesti-
heterotrimer composed of a 110- to 130-kDa myosin target- nal smooth muscle homogenates, we find no evidence for Zip
ing-binding subunit (MBS), a 37-kDa catalytic phosphatase kinase in the myometrial soluble fraction (S. Tichenor and

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subunit (PP1c), and a 20-kDa protein subunit of presently I. L. O. Buxton, unpublished observations).
unknown function (Hofmann et al., 2000) that may be in- Whether or not the Zip kinase is present, a large (indeed
volved with subcellular localization (Takizawa et al., 2003). A bewildering) number of putative MP kinase inhibitors are
large body of work has concentrated on MP, its mode of thought to prevent the activity of the PP1c phosphatase
regulation, and how the phosphatase can control smooth activity of MP, including ROK, CPI17 (MacDonald et al.,
muscle quiescence (for review, see Hartshorne et al., 1998). 2001b), integrin-linked kinase (Muranyi et al., 2002), PAK
Inhibition of MP is thought to contribute to Ca2⫹ sensitiza- (Takizawa et al., 2002a), Inhibitor-4 (Shirato et al., 2000),
tion, a phenomenon in which greater force is produced than and PPP1R14A (Li et al., 2001), to name a few. Although the
would result from elevation of Ca2⫹ and activation of MLCK matter is still controversial, phosphorylations of MP at var-
alone (Surks et al., 1999; Hofmann et al., 2000); this is a ious sites by a number of kinases result in decreased activity
striking aspect of contractile regulation in smooth muscle, of the PP1c (Kimura et al., 1996) as well as decreased binding
because its corollary is that MP activity alone reduces force of the MBS to myosin (Velasco et al., 2002). In particular,
generation (Kitazawa et al., 1991). CPI17 has recently been described in human myometrium,
The principal Ca2⫹-independent pathway thought to in- and its expression increased in tissue during pregnancy
crease force in smooth muscle is via inactivation of the phos- (Ozaki et al., 2003).
phatase activity of MP. This is thought to occur through Activation of the MP must therefore involve dephosphory-
activation of Rho kinase (ROK) by membrane anchored lation of the MBS at one or more of those sites phosphory-
RhoA-GTP. Active ROK has been shown to phosphorylate lated (Thr-695, Thr-850, and Thr-697) by inhibitory kinases.
MBS at Thr-695, Thr-850, or both, leading to inhibition of the In theory, when these sites are in a dephosphorylated state,
phosphatase and subsequent increase in MLC20 phosphory- MBS can interact with rMLC20, removing phosphates at
lation resulting in increased force without a Ca2⫹ increase Ser-18 and Ser-19, thus decreasing cross-bridge cycling and
(Feng et al., 1999). ROK is known to be activated by contrac- force generation. What then activates MP? In particular, how
tile agonists in smooth muscles (Somlyo and Somlyo, 2000) are the phosphorylations on the MBS reversed? Neither of
and to regulate expression of the smooth muscle contractile these questions has been answered in smooth muscle. In-
phenotype (Halayko and Solway, 2001). Although the inhib- deed, some suggest that MBS is not dephosphorylated signif-
itory phosphorylation sites on MBS have been suggested to icantly in smooth muscle (Takizawa et al., 2002b; Niiro et al.,
convey slight differences in the way they promote phospha- 2003), a notion that is not intellectually pleasing. Because
tase inhibition (Velasco et al., 2002), some investigators have MP is inhibited by phosphorylation of the MBS subunit by
shown that another pathway exists to regulate the phospha- kinases such as CPI17, it stands to reason that without
tase. dephosphorylation of these sites within the time frame of
Recently, a kinase thought to interact with its substrates contraction/relaxation, Ca2⫹ sensitization would be a perma-
based on the presence in their sequence of a leucine-rich nent on-switch and thus not reversibly measurable. Are we to
domain conferring a set of repeated bends (seven) has been believe that, once phosphorylated in the first moments of
described in nonmuscle cells (Murata-Hori et al., 1999). In function, the protein is never again to be without these in-
smooth muscle, the Zip-like kinase has been shown to be hibitory phosphorylations until replaced by new protein syn-
associated with the MP and to phosphorylate MBS at Thr- thesis? These data are hard to reconcile with the bulk of data
697, subsequently inhibiting the phosphatase, and thus pro- in smooth muscle showing the role of MBS phosphorylation
moting calcium sensitization (MacDonald et al., 2001a). A by kinases associated with contractile agonists (e.g., RhoA).
study appearing soon after this demonstrated that Zip kinase Regarding MP activation, we know that the PP1c is activated
directly phosphorylates rMLC20 on the same sites as MLCK by interaction with PKG. When PKG is activated by cGMP
but in a Ca2⫹-independent manner and that this, rather than elevation, the leucine zipper located on the N-terminal region
the phosphorylation of the MBS of MP, was the basis of the of PKG can interact with the leucine zipper on the C-terminal
effect of Zip kinase to enhance contraction (Niiro and Ikebe, region of MBS and signal rMLC20 dephosphorylation (Hof-
2001). Although this controversy is not yet resolved, it is mann et al., 2000). This interaction is not, however depen-
likely that both of these activities of the Zip kinase take place dent on PKG phosphorylation of MP, although that does
 A Biochemical Conundrum in Smooth Muscle 1055
occur (Nakamura et al., 1999). Indeed, the presence of a central region of the cell. PKG is also known to be colocalized
leucine zipper region in the MBS of MP is thought to be near the plasma membrane in smooth muscle cells (Koller et
consistent with the direct interaction of these two proteins al., 2003). It is possible, then, that the action of NO to relax
(Surks et al., 1999). myometrium is cGMP-independent, whereas the action of
Considering that global elevation of cGMP does not relax other inhibitory agonists is cGMP-dependent and restricted
myometrial smooth muscle, it is possible that the presence of to a particular region of the cell.
an isoform of MBS lacking the leucine zipper (MBSNZ) and
residing in the non lipid-raft/caveolar region of the cell ex-
plains this lack of a cGMP-mediated response. Such an iso-
KATP Channels
form is expressed in avian smooth muscle developmentally Although some data implicate KCa channels in the cGMP-
(Pfitzer et al., 1986; Khatri et al., 2001). The corollary we independent actions of NO in myometrium, there are reports
suggest for myometrium is that elevation of cGMP in the that the target of NO in myometrium is the KATP channel
lipid-raft/caveolar region of the myocyte and activation of instead (Modzelewska et al., 1998). Although mRNA has
PKG in that region (proposed to be PKG II; Fig. 2) activate been detected for this channel in human myometrium (Chien
MP by binding to an isoform of MBS containing the leucine et al., 1999; Curley et al., 2002), there is little (Hamada et al.,
zipper expressed and assembled with MP holoenzyme in this 1994) compelling electrophysiological evidence that KATP
region of the cell and resulting in activation of the phospha- channels are expressed there. This lack of evidence notwith-

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tase. Recently, Huang et al. (2004) showed that the binding of standing, it is possible that the channel exists in a closed
PKGI with MBS does not require the C-terminal zipper motif conformation unless the metabolic state of the tissue is com-
to be present in the MBS, whereas the dephosphorylation of promised or the channels are activated by an event such as
rMLC20 does. Although these data come from experiments in S-nitrosylation. Whatever the case, the hypothesis that KATP
cultured chicken gizzard smooth muscle cells in culture, they channels are present and mediate, in whole or part, the
offer an exciting context in which to consider the cGMP actions of NO to relax myometrium has yet to be ruled out.
insensitivity of myometrium. In myometrium, perhaps the What is different about myometrium? Might it be that
MBSNZ interacts with cGMP-PKGI accumulated after NO cGMP does not activate the cGMP-PK (PKG) known to be
action on the muscle. expressed in myometrium (Tamura et al., 1996; Word and
Consistent with such a notion, some investigators have Cornwell, 1998; Hennan and Diamond, 2001)? It has been
suggested that MP subunits are targeted to a region near the suggested that cGMP is ineffective in pregnant myometrium
plasma membrane in an agonist-specific fashion (Shin et al., because PKG is down-regulated (Word and Cornwell, 1998).
2002) such that the MBS subunit remains in the periphery of Although there may be changes in PKG expression in myo-
the cell while the PP1c phosphatase subunit is seen in the metrium, it is clear that cGMP elevation after NO donor

Fig. 2. Proposed compartmentation of signaling in the uterine smooth muscle myocyte. In cholesterol-rich (C), sphingolipid-rich (orange 䡬) regions
of the myocyte membrane (detergent-insoluble glycolipid-rich domains/caveolar microdomain), signaling proteins such as the particulate guanylyl
cyclase bind agonists such as uroguanylin (uG) and results in increased levels of cGMP locally. Cyclic GMP activates its cognate protein kinase (PKG)
that is localized in the region of the cGMP elevation by a G-kinase anchoring protein (GKAP) and permits the phosphorylation/interaction with the
MBS of myosin phosphatase (PP1) that dephosphorylates the regulatory myosin light chains (rMLC) releasing phosphate (F) and promoting relaxation
(‹) of actin-myosin interaction and thus relaxation of the muscle. Contractile agonists such as oxytocin (OT) stimulate G-protein regulated activation
of the kinase (RhoA) that phosphorylates MBS leading to inactivation of PP1. Other kinases, such as CPI 17, that may be activated by the
oxytocin-mediated rise in intracellular calcium (data not shown) also phosphorylate and inactivate PP1. The notion that all of these events are
organized in the signaling microdomain is illustrated by the presence of filamentous proteins (linked circles) that may contribute to localization of
signaling proteins.
1056 Buxton

stimulation of myometrium leads to protein phosphorylation otide phosphate (NAADP) is a recently discovered nucleotide
(Hennan and Diamond, 2001), just not relaxation. For exam- with intracellular Ca⫹2-releasing properties (Lee, 1994).
ple, the vasodilator associated protein VASP, a known PKG NAADP-induced Ca⫹2 release was first described in sea ur-
substrate in smooth muscle, is phosphorylated in response to chin egg homogenates (Chini et al., 1995) but has now been
cGMP elevation in myometrium (Tichenor et al., 2003). If described in mammalian cells (Yusufi et al., 2001a) and tis-
cGMP activates PKG, what might explain the apparent in- sues (for review, see Chini et al., 2002) including smooth
dependence of the NO-induced relaxation to global elevation muscle (Yusufi et al., 2002). Ca⫹2 release elicited by NAADP
of cGMP and activation of its associated kinase? differs in many ways from Ca⫹2 release controlled by cyclic
A partial answer is evident in a comparison of the actions ADP-ribose (the endogenous ryanodine receptor agonist) and
of the S-nitroso thiol NO donors and non-nitroso thiol com- inositol 1,4,5-trisphosphate (Genazzani and Billington,
pounds such as 3-morpholinosydnonimine (SIN-1). Although 2002). Properties of NAADP-induced calcium release include:
both of these agents cause significant global elevations in 1) the absence of regulation by Mg2⫹ and Ca2⫹; 2) NAADP-
cGMP in myometrium and other smooth muscles, in myome- induced Ca2⫹ release is fully desensitized by prior exposure
trium, only the S-nitroso compound, S-nitroso N-acetyl pen- to low concentrations of NAADP (Genazzani et al., 1996); and
icillamine (SNAP), relaxes the tissue once contracted by oxy- 3) the Ca2⫹ release induced by NAADP is insensitive to a
tocin (Buxton et al., 2001; Tichenor et al., 2003). These data wide range of changes in pH (Chini et al., 1998) and thus may
demonstrate again that global elevations of cGMP could not be active in microdomain environments. These characteris-
tics make NAADP a unique trigger of intracellular Ca⫹2

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be the mechanism through which NO signals relaxation of
myometrium. Based on work in airway smooth muscle, Jans- release/entry and suggest the possibility that this release
sen et al. (2000) proposed that SNAP, unlike SIN-1, caused pathway subserves the nitrosothiol-dependent release of
calcium release and that that was the basis for its action to Ca2⫹ in myometrium.
relax airway smooth muscle, whereas SIN-1 did not and thus Synthesis of NAADP by a base-exchange reaction has been
must work through cGMP. Although these authors did not described in several mammalian tissues, including brain,
actually measure cGMP in their study, the notion that an NO heart, liver, spleen, and kidney (Chini and Dousa, 1995;
donor might cause release of intracellular calcium was, if Cheng et al., 2001). Furthermore, it has also been reported
surprising, consistent with the notion that KCa channel acti- that ADP-ribosyl cyclase (CD38) is capable of catalyzing the
vation might be the basis of the action of NO in myometrium synthesis of NAADP in ‘smooth muscle-like’ mesangial cells
(Fig. 1). We have established in myometrium and in airway (Yusufi et al., 2001b). Although we are not aware of a de-
muscle that both of these donors cause global elevation of tailed description of NAADP synthesis or its receptor in
cGMP. To our surprise, SIN-1 was unable to relax myome- smooth muscle, CD38 is clearly expressed in myometrium
trium, whereas SNAP was quite effective. Here then was a (Dogan et al., 2002). It is possible that CD38 catalyzes the
correlation between relaxation and NO effects on intracellu- synthesis of NAADP in lipid-rich signaling domains of uter-
lar calcium release and a lack of correlation between global ine smooth muscle cells. This could occur as a result of
cGMP accumulation and relaxation (Tichenor et al., 2003). S-nitrosylation and activation of its synthesis via an effect on
The possibility also exists that SNAP was effective only be- CD38 or via its receptor; this effect could subserve mem-
cause it is an S-nitroso compound and able to S-nitrosylate brane-limited elevations in Ca2⫹ and constitute the cGMP-
critical myometrial substrates such as the KCa channel(s) or independent NO-mediated activation of KCa channels.
perhaps an as-yet-unknown substrate.
Caveolar Signaling
Leads from Genomic Studies Lipid-enriched signaling domains such as caveolae and
Genomic and proteomic studies of the physiology of partu- detergent-insoluble glycolipid-rich domains (Parton and Si-
rition have begun to shed light on the mechanisms of labor. mons, 1995) are recognized to play roles in many cellular
Several studies (Aguan et al., 2000; Chan et al., 2002; Bethin processes, especially in receptor signal-transduction (for re-
et al., 2003; Girotti and Zingg, 2003) seem particularly useful view, see Anderson, 1998), explaining, at least in part, early
in the context of understanding which changes might shed observations of subcellular compartmentation (Buxton and
light on the mechanisms of signaling regulation in the myo- Brunton, 1983) before caveolar signaling was known. Re-
metrium, and available work has been reviewed (Romero et cently, studies of the regulation of receptor signaling in en-
al., 2002). One of the genes that is very markedly up-regu- dothelial caveolae have suggested their role as calcium sen-
lated between day 0 and term in the rat is that encoding the sors (Kaiser et al., 2002). Caveolae are richly expressed in
particulate guanylyl cyclase (pGC) activator, uroguanylin smooth muscles, including myometrium (Kwan et al., 1986),
(Girotti and Zingg, 2003). Also up-regulated are transcripts and their numbers are regulated (Turi et al., 2001). Perhaps
for caveolin, consistent with the notion that signaling do- the conundrum presented here can be resolved on the basis of
mains develop in relation to the development of quiescence the organization of receptor signaling in myometrium be-
during gestation. tween detergent-insoluble glycolipid-rich domains, including
rafts and caveolae (Anderson and Jacobson, 2002), and the
soluble and myo-filamentous regions of the cell.
NAADP Releasable Ca2ⴙ Pool Some endogenous peptides that relax the myometrium
Release of calcium from intracellular stores is established may target the lipid-rich signaling domains of uterine
as a central component of numerous receptor-mediated sig- smooth muscle cells. Guanylin and uroguanylin are peptides
naling pathways and is obviously central to understanding (15 and 16 amino acids respectively) homologous to the heat-
the biology of smooth muscle. Nicotinic acid adenine dinucle- stable enterotoxin of Escherichia coli. These peptides were
 A Biochemical Conundrum in Smooth Muscle 1057
originally discovered in the gastrointestinal tract (Field et et al., 1995; Izumi and Garfield, 1995; Longo et al., 1999;
al., 1978; Hughes et al., 1978), where they regulate water and Vedernikov et al., 2000).
electrolyte balance through a cGMP-dependent mechanism.
Enterotoxigenic strains of bacteria produce heat-stable ente- Conclusions
rotoxins that stimulate chloride secretion leading to accumu-
lation in the gastrointestinal lumen and subsequent secre- Less is currently known about the contractile regulation of
tory diarrhea. In the 1980s, specific binding sites for heat myometrial smooth muscle than other smooth muscles. Ex-
stable enterotoxin peptides were discovered (membrane isting evidence suggests that the relaxation of uterine
bound guanylyl cyclase) in intestine and other tissues such as smooth muscle by NO and NO donors does not result from
kidney and lung (Forte et al., 1988, 1989). The discovery of elevation of cGMP in the muscle. This disassociation of cGMP
receptors (pGC) in tissues not thought to be exposed to STa accumulation and relaxation of the muscle suggests interest-
peptides suggested that endogenous agonists might exist. ing possibilities for both NO signaling and the regulation of
The first of these to be discovered was guanylin (Currie et al., contractile protein interactions in uterine muscle. In the case
1992). Two other members of the family are also known in of NO signaling, myometrial smooth muscle may provide an
human (Nakazato, 2001), uroguanylin and lymphoguanylin, opportunity to examine cGMP-independent signaling such as
the latter of which will not be considered here. Prouroguany- S-nitrosylation of proteins that may be involved in regional
lin and proguanylin proteins are believed to be produced by increases in calcium and/or direct activation of potassium
channels. Because cGMP accumulation and activation of

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enterochromaffin (Perkins et al., 1997) and endocrine cells
(Magert et al., 1998) in the intestine and differentiated epi- PKG is expected to relax smooth muscle in large measure via
thelial cells in the kidney (Nakazato et al., 1998). These PKG activation of myosin phosphatase, the failure of cGMP
precursor proteins are inactive and circulate in the blood- accumulation to explain the actions of NO in uterine muscle
stream (Beltowski, 2001). Little is known about the actual suggests fundamental differences in the regulation of myosin
proteolytic generation of the peptides guanylin and urogua- phosphatase in myometrium.
nylin from their pro-forms, although chymotrypsin may be Examining these possibilities in the context of regional
responsible for production of guanylin peptides in the gut signaling domains within the uterine smooth muscle cell,
(Magert et al., 1998). The fact that there is only a 20% together with the actions of unique peptides such as urogua-
identity in the sequences of human guanylin and uroguany- nylin, offers an interesting framework in which to consider
lin suggests that their roles may be different. existing data and plan future work.
We suggest that uroguanylin and not guanylin is produced
in uterine glandular cells during pregnancy to signal to myo- Acknowledgments
metrium in a paracrine fashion and maintain uterine quies- I am grateful to Stephen Tichenor for his contributions to the work
cence. This hypothesis is supported by the findings that discussed and for critical review of the manuscript.
prouroguanylin and proguanylin are the products of different
genes (Magert et al., 1998); uroguanylin, not guanylin, gene References
expression is up-regulated in uterine tissue during preg- American College of Obstetricians and Gynecologists Committee on Practice Bulle-
tins (2003) ACOG Practice Bulletin. Clinical management guidelines for obstetri-
nancy (Girotti and Zingg, 2003). Perhaps most interestingly, cian-gynecologist. Number 43, May 2003. Management of preterm labor. Obstet
uroguanylin is thought to be 100 times more potent than Gynecol 101:1039 –1047.
Aguan K, Carvajal JA, Thompson LP, and Weiner CP (2000) Application of a
guanylin in an acidic environment (Hamra et al., 1997). Our functional genomics approach to identify differentially expressed genes in human
hypothesis that uroguanylin acts at the myocyte caveolae is myometrium during pregnancy and labour. Mol Hum Reprod 6:1141–1145.
Ahern GP, Hsu SF, and Jackson MB (1999) Direct actions of nitric oxide on rat
supported both by the presence of pGC activity in myome- neurohypophysial K⫹ channels. J Physiol (Lond) 520:165–176
trium (Buhimschi et al., 2000; Telfer et al., 2001) and by the Ahern GP, Klyachko VA, and Jackson MB (2002) CGMP and S-nitrosylation: two
routes for modulation of neuronal excitability by NO. Trends Neurosci 25:510 –517.
notion that the receptor environment in the caveolar cleft Anderson RG (1998) The caveolae membrane system. Annu Rev Biochem 67:199 –
may be acidic because of the concentration of acidic lipids 225.
Anderson RG and Jacobson K (2002) A role for lipid shells in targeting proteins to
(Anderson, 1998). caveolae, rafts and other lipid domains. Science (Wash DC) 296:1821–1825.
This hypothesis regarding the origins and actions of Bao S, Rai J, and Schreiber J (2002) Expression of nitric oxide synthase isoforms in
human pregnant myometrium at term. J Soc Gynecol Investig 9:351–356.
uroguanylin on myometrium is also supported by data from Bartlett SR, Bennett PR, Campa JS, Dennes WJ, Slater DM, Mann GE, Poston L,
other labs (Weiner et al., 1994; Buhimschi et al., 2000; Car- and Poston R (1999) Expression of nitric oxide synthase isoforms in pregnant
human myometrium. J Physiol 521:705–716.
vajal et al., 2001; Fulep et al., 2001), although others may Beltowski J (2001) Guanylin and related peptides. J Physiol Pharmacol 52:351–375.
interpret the data differently. It is noteworthy that Weiner et Benkusky NA, Fergus DJ, Zucchero TM, and England SK (2000) Regulation of the
al. (1994) showed that uterine cGMP is elevated over preg- Ca2⫹-sensitive domains of the maxi-K channel in the mouse myometrium during
gestation. J Biol Chem 275:27712–27719.
nancy toward term and that this accumulation of the second Bethin KE, Nagai Y, Sladek R, Asada M, Sadovsky Y, Hudson TJ, and Muglia LJ
messenger is not caused by NO activity. Although these (2003) Microarray analysis of uterine gene expression in mouse and human preg-
nancy. Mol Endocrinol 17:1454 –1469.
authors do not favor a role for pGC in this elevation (Carvajal Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, and Cohen RA (1994) Nitric oxide
et al., 2001), their study of natriuretic peptide-induced relax- directly activates calcium-dependent potassium channels in vascular smooth mus-
cle. Nature (Lond) 368:850 – 853.
ation of oxytocin-stimulated myometrium in the guinea pig Bradley KK, Buxton IL, Barber JE, McGaw T, and Bradley ME (1998) Nitric oxide
did not test the effects of uroguanylin. Studies by Buhimschi relaxes human myometrium by a cGMP-independent mechanism. Am J Physiol
275:C1668 –C1673.
et al. (2000) and Fulep et al. (2001) demonstrate relaxant Brune B, Mohr S, and Messmer UK (1996) Protein thiol modification and apoptotic
effects of natriuretic peptides on uterine contractions and a cell death as cGMP-independent nitric oxide (NO) signaling pathways. Rev Physiol
Biochem Pharmacol 127:1–30:1–30.
lack of effect of cGMP elevations from sGC activation. These Buhimschi I, Yallampalli C, Dong YL, and Garfield RE (1995) Involvement of a nitric
data are useful considering previous contributions asserting oxide-cyclic guanosine monophosphate pathway in control of human uterine con-
tractility during pregnancy. Am J Obstet Gynecol 172:1577–1584.
that NO relaxes myometrium in a cGMP-dependent fashion Buhimschi IA, Martin-Clark O, Aguan K, Thompson LP, and Weiner CP (2000)
(Izumi et al., 1993; Yallampalli et al., 1993, 1994; Buhimschi Differential alterations in responsiveness in particulate and soluble guanylate
1058 Buxton

cyclases in pregnant guinea pig myometrium. Am J Obstet Gynecol 183:1512– intestinal uroguanylin/guanylin receptor-mediated responses by mucosal acidity.
1519. Proc Natl Acad Sci USA 94:2705–2710.
Buxton IL and Brunton LL (1983) Compartments of cyclic AMP and protein kinase Hartshorne DJ, Ito M, and Erdodi F (1998) Myosin light chain phosphatase: subunit
in mammalian cardiomyocytes. J Biol Chem 258:10233–10239. composition, interactions and regulation. J Muscle Res Cell Motil 19:325–341.
Buxton IL, Crow W, and Mathew SO (2000) Regulation of uterine contraction: Hennan JK and Diamond J (1998) Evidence that spontaneous contractile activity in
mechanisms in preterm labor. AACN Clin Issues 11:271–282. the rat myometrium is not inhibited by NO-mediated increases in tissue levels of
Buxton IL, Kaiser RA, Malmquist NA, and Tichenor S (2001) NO-induced relaxation cyclic GMP. Br J Pharmacol 123:959 –967.
of labouring and non-labouring human myometrium is not mediated by cyclic Hennan JK and Diamond J (2001) Effect of NO donors on protein phosphorylation in
GMP. Br J Pharmacol 134:206 –214. intact vascular and nonvascular smooth muscles. Am J Physiol 280:H1565–
Carvajal JA, Aguan K, Thompson LP, Buhimschi IA, and Weiner CP (2001) Natri- H1580.
uretic peptide-induced relaxation of myometrium from the pregnant guinea pig is Hofmann F, Ammendola A, and Schlossmann J (2000) Rising behind NO: cGMP-
not mediated by guanylate cyclase activation. J Pharmacol Exp Ther 297:181–188. dependent protein kinases. J Cell Sci 113:1671–1676.
Chan EC, Fraser S, Yin S, Yeo G, Kwek K, Fairclough RJ, and Smith R (2002) Huang QQ, Fisher SA, and Brozovich FV (2004) Unzipping the role of myosin light
Human myometrial genes are differentially expressed in labor: a suppression chain phosphatase in smooth muscle cell relaxation J Biol Chem 279:597– 603.
subtractive hybridization study. J Clin Endocrinol Metab 87:2435–2441. Hughes JM, Murad F, Chang B, and Guerrant RL (1978) Role of cyclic GMP in the
Chanrachakul B, Matharoo-Ball B, Turner A, Robinson G, Broughton-Pipkin F, action of heat-stable enterotoxin of Escherichia coli. Nature (Lond) 271:755–756.
Arulkumaran S, and Khan RN (2003) Immunolocalization and protein expression Izumi H and Garfield RE (1995) Relaxant effects of nitric oxide and cyclic GMP on
of the alpha subunit of the large-conductance calcium-activated potassium channel pregnant rat uterine longitudinal smooth muscle. Eur J Obstet Gynecol Reprod
in human myometrium 1. Reproduction 126:43– 48. Biol 60:171–180.
Cheng J, Yusufi AN, Thompson MA, Chini EN, and Grande JP (2001) Nicotinic acid Izumi H, Yallampalli C, and Garfield RE (1993) Gestational changes in L-arginine-
adenine dinucleotide phosphate: a new Ca2⫹ releasing agent in kidney. J Am Soc induced relaxation of pregnant rat and human myometrial smooth muscle. Am J
Nephrol 12:54 – 60. Obstet Gynecol 169:1327–1337.
Chien EK, Zhang Y, Furuta H, and Hara M (1999) Expression of adenosine triphos- Janssen LJ, Premji M, Lu-Chao H, Cox G, and Keshavjee S (2000) NO⫹ but not NO
phate-sensitive potassium channel subunits in female rat reproductive tissues: radical relaxes airway smooth muscle via cGMP-independent release of internal
overlapping distribution of messenger ribonucleic acid for weak inwardly rectify- Ca2⫹. Am J Physiol 278:L899 –L905.

Downloaded from molpharm.aspetjournals.org at ASPET Journals on April 22, 2015

ing potassium channel subunit 6.1 and sulfonylurea-binding regulatory subunit 2. Jia L, Bonaventura C, Bonaventura J, and Stamler JS (1996) S-Nitrosohaemoglobin:
Am J Obstet Gynecol 180:1121–1126. a dynamic activity of blood involved in vascular control. Nature (Lond) 380:221–
Chini EN, Beers KW, and Dousa TP (1995) Nicotinate adenine dinucleotide phos- 226.
phate (NAADP) triggers a specific calcium release system in sea urchin eggs. Jones GD and Poston L (1997) The role of endogenous nitric oxide synthesis in
J Biol Chem 270:3216 –3223. contractility of term or preterm human myometrium. Br J Obstet Gynaecol 104:
Chini EN, Chini CC, Kato I, Takasawa S, and Okamoto H (2002) CD38 is the major 241–245.
enzyme responsible for synthesis of nicotinic acid-adenine dinucleotide phosphate Jones KA, Lorenz RR, Warner DO, Katusic ZS, and Sieck GC (1994) Changes in
in mammalian tissues. Biochem J 362:125–130. cytosolic cGMP and calcium in airway smooth muscle relaxed by 3-morpholinosyd-
Chini EN and Dousa TP (1995) Enzymatic synthesis and degradation of nicotinate nonimine. Am J Physiol 266:L9 –L16.
adenine dinucleotide phosphate (NAADP), a Ca2⫹-releasing Agonist, in rat tis- Kaczorowski GJ, Knaus HG, Leonard RJ, McManus OB, and Garcia ML (1996)
sues. Biochem Biophys Res Commun 209:167–174. High-conductance calcium-activated potassium channels; structure, pharmacology
Chini EN, Liang M, and Dousa TP (1998) Differential effect of pH upon cyclic-ADP- and function. J Bioenerg Biomembr 28:255–267.
ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2⫹ release sys- Kaiser RA, Oxhorn BC, Andrews G, and Buxton IL (2002) Functional compartmen-
tems. Biochem J 335:499 –504. tation of endothelial P2Y receptor signaling. Circ Res 91:292–299.
Curley M, Cairns MT, Friel AM, McMeel OM, Morrison JJ, and Smith TJ (2002) Khan RN, Matharoo-Ball B, Arulkumaran S, and Ashford ML (2001) Potassium
Expression of mRNA transcripts for ATP-sensitive potassium channels in human channels in the human myometrium. Exp Physiol 86:255–264.
myometrium. Mol Hum Reprod 8:941–945. Khan RN, Smith SK, Morrison JJ, and Ashford ML (1993) Properties of large-
Currie MG, Fok KF, Kato J, Moore RJ, Hamra FK, Duffin KL, and Smith CE (1992) conductance K⫹ channels in human myometrium during pregnancy and labour.
Guanylin: an endogenous activator of intestinal guanylate cyclase. Proc Natl Acad Proc R Soc Lond B Biol Sci 251:9 –15.
Sci USA 89:947–951. Khatri JJ, Joyce KM, Brozovich FV, and Fisher SA (2001) Role of myosin phospha-
Davis KL, Martin E, Turko IV, and Murad F (2001) Novel Effects of Nitric Oxide. tase isoforms in cGMP-mediated smooth muscle relaxation. J Biol Chem 276:
Annu Rev Pharmacol Toxicol 41:203–236. 37250 –37257.
Diamond J (1983) Lack of correlation between cyclic GMP elevation and relaxation Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng
of nonvascular smooth muscle by nitroglycerin, nitroprusside, hydroxylamine and J, Nakano T, Okawa K, et al. (1996) Regulation of myosin phosphatase by rho and
sodium azide. J Pharmacol Exp Ther 225:422– 426. rho-associated kinase (rho-kinase). Science (Wash DC) 273:245–248.
Dogan S, White TA, Deshpande DA, Murtaugh MP, Walseth TF, and Kannan MS Kishnani NS and Fung HL (1996) Nitric oxide generation from pharmacological
(2002) Estrogen increases CD38 gene expression and leads to differential regula- nitric oxide donors. Methods Enzymol 268:259 –265.
tion of adenosine diphosphate (ADP)-ribosyl cyclase and cyclic ADP-ribose hydro- Kitazawa T, Gaylinn BD, Denney GH, and Somlyo AP (1991) G-protein-mediated
lase activities in rat myometrium. Biol Reprod 66:596 – 602. Ca2⫹ sensitization of smooth muscle contraction through myosin light chain phos-
Duckitt K and Thornton S (2002) Nitric oxide donors for the treatment of preterm phorylation. J Biol Chem 266:1708 –1715.
labour. Cochrane Database Syst Rev CD002860. Klyachko VA, Ahern GP, and Jackson MB (2001) cGMP-mediated facilitation in
Eckman DM, Weinert JS, Buxton ILO, and Keef KD (1994) Cyclic GMP independent nerve terminals by enhancement of the spike after hyperpolarization. Neuron
relaxation and hyperpolarization with acetylcholine in guinea pig coronary artery. 31:1015–1025.
Br J Pharmacol 111:1053–1060. Koller A, Schlossmann J, Ashman K, Uttenweiler-Joseph S, Ruth P, and Hofmann F
Ekerhovd E, Brannstrom M, Delbro D, and Norstrom A (1998) Nitric oxide mediated (2003) Association of phospholamban with a cGMP kinase signaling complex.
inhibition of contractile activity in the human uterine cervix. Mol Hum Reprod Biochem Biophys Res Commun 300:155–160.
4:915–920. Kuenzli KA, Bradley ME, and Buxton IL (1996) Cyclic GMP-independent effects of
Feng J, Ito M, Ichikawa K, Isaka N, Nishikawa M, Hartshorne DJ, and Nakano T nitric oxide on guinea-pig uterine contractility. Br J Pharmacol 119:737–743.
(1999) Inhibitory phosphorylation site for rho-associated kinase on smooth muscle Kuenzli KA, Buxton IL, and Bradley ME (1998) Nitric oxide regulation of monkey
myosin phosphatase. J Biol Chem 274:37385–37390. myometrial contractility. Br J Pharmacol 124:63– 68.
Field M, Graf LH Jr, Laird WJ, and Smith PL (1978) Heat-stable enterotoxin of Kwan CY, Berezin I, Daniel EE, and Lee RM (1986) Dense-cored membrane struc-
Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concen- tures in rat myometrial smooth muscle cells: increase in number during parturi-
tration and ion transport in small intestine. Proc Natl Acad Sci USA 75:2800 – tion. Can J Physiol Pharmacol 64:1219 –1222.
2804. Lander HM, Hajjar DP, Hempstead BL, Mirza UA, Chait BT, Campbell S, and
Forte LR, Krause WJ, and Freeman RH (1988) Receptors and cGMP signalling Quilliam LA (1997) A molecular redox switch on P21(Ras). Structural basis for the
mechanism for E. coli enterotoxin in opossum kidney. Am J Physiol 255:F1040 – nitric oxide-P21(Ras) interaction. J Biol Chem 272:4323– 4326.
F1046. Lang RJ, Harvey JR, McPhee GJ, and Klemm MF (2000) Nitric oxide and thiol
Forte LR, Krause WJ, and Freeman RH (1989) Escherichia coli enterotoxin recep- reagent modulation of Ca2⫹-activated K⫹ (BKCa) channels in myocytes of the
tors: localization in opossum kidney, intestine and testis. Am J Physiol 257:F874 – guinea-pig taenia caeci. J Physiol 525: 363–376.
F881. Lee HC (1994) Cyclic ADP-ribose: a calcium mobilizing metabolite of NAD⫹. Mol Cell
Fulep E, Vedernikov Y, Saade GR, and Garfield RE (2001) Contractility of late Biochem 138:229 –235.
pregnant rat myometrium is refractory to activation of soluble but not particulate Lees C, Campbell S, Jauniaux E, Brown R, Ramsay B, Gibb D, Moncada S, and
guanylate cyclase. Am J Obstet Gynecol 185:158 –162. Martin JF (1994) Arrest of preterm labour and prolongation of gestation with
Genazzani AA and Billington RA (2002) NAADP: an atypical Ca2⫹-release messen- glyceryl trinitrate, a nitric oxide donor. Lancet 343:1325–1326.
ger? Trends Pharmacol Sci 23:165–167. Li Z, Yu L, Zhang Y, Gao J, Zhang P, Wan B, Chen C, and Zhao S (2001) Identifi-
Genazzani AA, Empson RM, and Galione A (1996) Unique inactivation properties of cation of human, mouse and rat PPP1R14A, protein phosphatase-1 inhibitor
NAADP-sensitive Ca2⫹ release. J Biol Chem 271:11599 –11602. subunit 14A, & mapping human PPP1R14A to chromosome 19q13.13-Q13.2. Mol
Girotti M and Zingg HH (2003) Gene expression profiling of rat uterus at different Biol Rep 28:91–101.
stages of parturition. Endocrinology 144:2254 –2265. Longo M, Jain V, Vedernikov YP, Saade GR, Goodrum L, Facchinetti F, and Garfield
Halayko AJ and Solway J (2001) Molecular mechanisms of phenotypic plasticity in RE (1999) Effect of nitric oxide and carbon monoxide on uterine contractility
smooth muscle cells. J Appl Physiol 90:358 –368. during human and rat pregnancy. Am J Obstet Gynecol 181:981–988.
Hamada Y, Nakaya Y, Hamada S, Kamada M, and Aono T (1994) Activation of K⫹ MacDonald JA, Borman MA, Muranyl A, Somlyo A, Hartshorne DJ, and Hayward
channels by ritodrine hydrochloride in uterine smooth muscle cells from pregnant BS (2001a) identification of the endogenous smooth muscle myosin phosphatase-
women. Eur J Pharmacol 288:45–51. associated kinase. Proc Natl Acad Sci USA 98:2419 –2424.
Hamra FK, Eber SL, Chin DT, Currie MG, and Forte LR (1997) Regulation of MacDonald JA, Eto M, Borman MA, Brautigan DL, and Haystead TA (2001b) Dual
 A Biochemical Conundrum in Smooth Muscle 1059
Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin and phosphatase, Ca2⫹ current in glomus cells of the rabbit carotid body via a cGMP-independent
by MYPT-associated kinase. FEBS Lett 493:91–94. mechanism. J Neurophysiol 81:1449 –1457.
Magert HJ, Reinecke M, David I, Raab HR, Adermann K, Zucht HD, Hill O, Hess R, Surks HK, Mochizuki N, Kasai Y, Georgescu SP, Tang KM, Ito M, Lincoln TM, and
and Forssmann WG (1998) Uroguanylin: gene structure, expression, processing as Mendelsohn ME (1999) Regulation of myosin phosphatase by a specific interaction
a peptide hormone and co-storage with somatostatin in gastrointestinal D-cells. with cGMP-dependent protein kinase Ialpha. Science (Wash DC) 286:1583–1587.
Regul Pept 73:165–176. Takizawa N, Koga Y, and Ikebe M (2002a) Phosphorylation of CPI17 and myosin
Mazzone JN, Kaiser RA, and Buxton IL (2002) Calcium-activated potassium channel binding subunit of type 1 protein phosphatase by P21-activated kinase. Biochem
expression in human myometrium: effect of pregnancy. Proc West Pharmacol Soc Biophys Res Commun 297:773–778.
45:184 –186. Takizawa N, Niiro N, and Ikebe M (2002b) Dephosphorylation of the two regulatory
Mazzuco TL, Andre E, and Calixto JB (2000) Contribution of nitric oxide, prostanoids components of myosin phosphatase, MBS and CPI17. FEBS Lett 515:127–132.
and Ca2⫹-activated K⫹ channels to the relaxant response of bradykinin in the Takizawa N, Schmidt DJ, Mabuchi K, Villa-Moruzzi E, Tuft RA, and Ikebe M (2003)
guinea pig bronchus in vitro. Naunyn Schmiedeberg’s Arch Pharmacol 361:383– M20, the small subunit of PP1M, binds to microtubules. Am J Physiol 284:C250 –
390. C262.
McCarron JG, Bradley KN, and Muir TC (2002) Ca2⫹ signalling and Ca2⫹-activated Tamura N, Itoh H, Ogawa Y, Nakagawa O, Harada M, Chun TH, Suga S, Yoshimasa
K⫹ channels in smooth muscle. Novartis Found Symp 246:52– 64. T, and Nakao K (1996) cDNA cloning and gene expression of human type Ialpha
Modzelewska B, Sipowicz MA, Saavedra JE, Keefer LK, and Kostrzewska A (1998) cGMP-dependent protein kinase. Hypertension 27:552–557.
Involvement of K⫹ ATP channels in nitric oxide-induced inhibition of spontaneous Telfer JF, Itoh H, Thomson AJ, Norman JE, Nakao K, Campa JS, Poston L, Tribe
contractile activity of the nonpregnant human myometrium. Biochem Biophys Res RM, and Magness RR (2001) Activity and expression of soluble and particulate
Commun 253:653– 657. guanylate cyclases in myometrium from nonpregnant and pregnant women: down-
Muranyi A, MacDonald JA, Deng JT, Wilson DP, Haystead TA, Walsh MP, Erdodi F, regulation of soluble guanylate cyclase at term. J Clin Endocrinol Metab 86:5934 –
Kiss E, Wu Y, and Hartshorne DJ (2002) Phosphorylation of the myosin phospha- 5943.
tase target subunit by integrin-linked kinase. Biochem J 366:211–216. Tiboni GM, Giampietro F, and Lamonaca D (2001) The soluble guanylate cyclase
Murata-Hori M, Suizu F, Iwasaki T, Kikuchi A, and Hosoya H (1999) ZIP kinase inhibitor methylene blue evokes preterm delivery and fetal growth restriction in a
identified as a novel myosin regulatory light chain kinase in HeLa cells. FEBS Lett mouse model. In Vivo 15:333–337.
451:81– 84. Tichenor SD, Malmquist NA, and Buxton IL (2003) Dissociation of cGMP accumu-

Downloaded from molpharm.aspetjournals.org at ASPET Journals on April 22, 2015

Nakamura M, Ichikawa K, Ito M, Yamamori B, Okinaka T, Isaka N, Yoshida Y, lation and relaxation in myometrial smooth muscle: effects of S-nitroso-N-
Fujita S, and Nakano T (1999) Effects of the phosphorylation of myosin phospha- acetylpenicillamine and 3-morpholinosyndonimine. Cell Signal 15:763–772.
tase by cyclic GMP-dependent protein kinase. Cell Signal 11:671– 676. Trottier G, Triggle CR, O’Neill SK, and Loutzenhiser R (1998) Cyclic GMP-
Nakazato M (2001) Guanylin family: new intestinal peptides regulating electrolyte dependent and cyclic GMP-independent actions of nitric oxide on the renal afferent
and water homeostasis. J Gastroenterol 36:219 –225. arteriole. Br J Pharmacol 125:563–569.
Nakazato M, Yamaguchi H, Date Y, Miyazato M, Kangawa K, Goy MF, Chino N, and Turi A, Kiss AL, and Mullner N (2001) Estrogen down-regulates the number of
Matsukura S (1998) Tissue distribution, cellular source and structural analysis of caveolae and the level of caveolin in uterine smooth muscle. Cell Biol Int 25:785–
rat immunoreactive uroguanylin. Endocrinology 139:5247–5254. 794.
Niiro N and Ikebe M (2001) Zipper-interacting protein kinase induces Ca2⫹-free Vedernikov YP, Syal AS, Okawa T, Jain V, Saade GR, and Garfield RE (2000) The
smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem role of cyclic nucleotides in the spontaneous contractility and responsiveness to
276:29567–29574. nitric oxide of the rat uterus at midgestation and term. Am J Obstet Gynecol
Niiro N, Koga Y, and Ikebe M (2003) Agonist-induced changes in the phosphorylation 182:612– 619.
of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two Velasco G, Armstrong C, Morrice N, Frame S, and Cohen P (2002) Phosphorylation
regulatory factors of myosin light chain phosphatase, in smooth muscle. Biochem of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850
J 369:117–128. induces its dissociation from myosin. FEBS Lett 527:101–104.
Ozaki H, Yasuda K, Kim YS, Egawa M, Kanzaki H, Nakazawa H, Hori M, Seto M, Vince GS, Starkey PM, Jackson MC, Sargent IL, and Redman CWG (1990) Flow
and Karaki H (2003) Possible role of the protein kinase C/CPI-17 pathway in the
cytometric characterisation of cell populations in human pregnancy decidua and
augmented contraction of human myometrium after gestation. Br J Pharmacol
isolation of decidual macrophages. J Immunol Methods 132:181–189.
140:1303–1312.
Weiner CP, Knowles RG, Nelson SE, and Stegink LD (1994) Pregnancy increases
Parton RG and Simons K (1995) Digging into caveolae. Science (Wash DC) 269:1398 –
guanosine 3⬘,5⬘-monophosphate in the myometrium independent of nitric oxide
1399.
synthesis. Endocrinology 135:2473–2478.
Perkins A, Goy MF, and Li Z (1997) Uroguanylin is expressed by enterochromaffin
White RE (1999) Cyclic GMP and ion channel regulation. Adv Second Messenger
cells in the rat gastrointestinal tract. Gastroenterology 113:1007–1014.
Phosphoprotein Res 33:251–277.
Pfitzer G, Merkel L, Ruegg JC, and Hofmann F (1986) Cyclic GMP-dependent
White RE, Lee AB, Shcherbatko AD, Lincoln TM, Schonbrunn A, and Armstrong DL
protein kinase relaxes skinned fibers from guinea pig taenia coli but not from
(1993) Potassium channel stimulation by natriuretic peptides through cGMP-
chicken gizzard. Pflueg Arch Eur J Physiol 407:87–91.
dependent dephosphorylation. Nature (Lond) 361:263–266.
Pinilla L, Gonzalez D, Tena-Sempere M, and Aguilar E (1998) Nitric oxide (NO)
Word RA, Casey ML, Kamm KE, and Stull JT (1991) Effects of cGMP on [Ca2⫹]i,
stimulates gonadotropin secretion in vitro through a calcium-dependent, cGMP-
Myosin light chain phosphorylation and contraction in human myometrium. Am J
independent mechanism. Neuroendocrinology 68:180 –186.
Physiol 260:C861–C867.
Romero R, Kuivaniemi H, and Tromp G (2002) Functional genomics and proteomics
in term and preterm parturition. J Clin Endocrinol Metab 87:2431–2434. Word RA and Cornwell TL (1998) Regulation of cGMP-induced relaxation and
Sanyal M, Nag TC, and Das C (2000) Localization of nitric oxide synthase in human cGMP-dependent protein kinase in rat myometrium during pregnancy. Am J
trophoblast cells: role of nitric oxide in trophoblast proliferation and differentia- Physiol 274:C748 –C756.
tion. Am J Reprod Immunol 43:70 –77. Yallampalli C, Garfield RE, and Byam-Smith M (1993) Nitric oxide inhibits uterine
Shimano M, Nakaya Y, Fukui R, Kamada M, Hamada Y, Maeda K, and Aono T contractility during pregnancy but not during delivery. Endocrinology 133:1899 –
(2000) Activation of Ca2⫹-activated K⫹ channels in human myometrium by nitric 1902.
oxide. Gynecol Obstet Invest 49:249 –254. Yallampalli C, Izumi H, Byam-Smith M, and Garfield RE (1994) An L-arginine-nitric
Shin HM, Je HD, Gallant C, Tao TC, Hartshorne DJ, Ito M, and Morgan KG (2002) oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits
Differential association and localization of myosin phosphatase subunits during contractility during pregnancy. Am J Obstet Gynecol 170:175–185.
agonist-induced signal transduction in smooth muscle. Circ Res 90:546 –553. Yu M, Sun CW, Maier KG, Harder DR, and Roman RJ (2002) Mechanism of cGMP
Shirato H, Shima H, Sakashita G, Nakano T, Ito M, Lee EY, and Kikuchi K (2000) contribution to the vasodilator response to NO in rat middle cerebral arteries.
Identification and characterization of a novel protein inhibitor of type 1 protein Am J Physiol 282:H1724 –H1731.
phosphatase. Biochemistry 39:13848 –13855. Yusufi AN, Cheng J, Thompson MA, Burnett JC, and Grande JP (2002) Differential
Somlyo AP and Somlyo AV (2000) Signal transduction by G-Proteins, rho-kinase and mechanisms of Ca2⫹ release from vascular smooth muscle cell microsomes. Exp
protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522 Pt Biol Med 227:36 – 44.
2:177–185. Yusufi AN, Cheng J, Thompson MA, Chini EN, and Grande JP (2001a) Nicotinic
Stamler JS, Singel DJ, and Loscalzo J (1992) Biochemistry of nitric oxide and its acid-adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2⫹
redox-activated forms. Science (Wash DC) 258:1898 –1902. release from mammalian cells. Biochem J 353:531–536.
Stamler JS, Toone EJ, Lipton SA, and Sucher NJ (1997) (S)NO signals: transloca- Yusufi AN, Cheng J, Thompson MA, Dousa TP, Warner GM, Walker HJ, and Grande
tion, regulation and a consensus motif. Neuron 18:691– 696. JP (2001b) CADP-ribose/ryanodine channel/Ca2⫹-release signal transduction
Steinberg SF and Brunton LL (2001) Compartmentation of G protein-coupled sig- pathway in mesangial cells. Am J Physiol 281:F91–F102.
naling pathways in cardiac myocytes. Annu Rev Pharmacol Toxicol 41:751–773.
Stull JT, Tansey MG, Word RA, Kubota Y, and Kamm KE (1991) Myosin light-chain Address correspondence to: Iain L. O. Buxton, Professor of Pharmacology,
kinase phosphorylation: regulation of the ca2⫹ sensitivity of contractile elements. University of Nevada School of Medicine MS318, 1664 N. Virginia Street,
Adv Exp Med Biol 304:129 –138. Reno, NV 89557. E-mail: [email protected]
Summers BA, Overholt JL, and Prabhakar NR (1999) Nitric Oxide Inhibits L-type