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Biology of Reproduction 64, 857-864 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Regulation of Cyclic Guanosine Monophosphate-Dependent Protein Kinase in Human Uterine Tissues During the Menstrual Cycle1

Trudy L. Cornwell2,a, Jie Lia, Hassan Sellaka, Primal de Lanerolleb, William H. Rodgersc, Rodney T. Millerd, and R. Ann Worde

a Department of Pathology, b Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019 Department of Physiology and Biophysics, c University of Illinois at Chicago, Chicago, Illinois 60612-7342 Department of Pathology, d Oregon Health Sciences University, Portland, Oregon 97201-3098 ProPath Laboratory, Inc., e Dallas, Texas 75207-4009 Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9032

ABSTRACT

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

cGMP, kinases, menstrual cycle, uterus

INTRODUCTION

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. At the end of the menstrual cycle, in the absence of embryo implantation, changes in endometrial and myometrial cells occur. An increase in myometrial contractility aids in the constriction of the spiral arterioles that regulate endometrial blood flow, with consequent tissue ischemia, endometrial disintegration, and bleeding [1]. Uterine contractility is clearly under the control of estrogen and progesterone [2] and varies with the menstrual cycle [1] and pregnancy [3].

The myometrium, consisting largely of phasic smooth muscle, contracts in response to an increase in intracellular calcium, induced by influx from external stores or release from intracellular pools. No single cellular mediator has been identified as a uterine smooth muscle relaxant, suggesting that multiple mechanisms may operate to control uterine smooth muscle contractility. Prostacyclin, nitric oxide (NO), and cyclic nucleotides have been identified as potential mediators of uterine relaxation [3–6].

Several cell types in the uterine compartment are capable of NO synthesis, as indicated by the expression of NO synthase isoforms and the measurement of NO metabolites [7]. Nitric oxide in nanomolar concentrations binds to soluble guanylate cyclase to catalyze cGMP formation. In addition, natriuretic peptides may activate the particulate isoform of guanylate cyclase resulting in increased levels of the cyclic nucleotide. Activation of the NO-cGMP signaling pathway results in the relaxation of several smooth muscle types. In vascular smooth muscle, the downstream target of this pathway essential for decreasing cytoplasmic calcium and tone is the cGMP-dependent protein kinase (PKG) [5, 6, 8]. Smooth muscle relaxation is brought about by the phosphorylation of substrates involved in calcium regulation and ion transport [5, 8–11]. Although all components of the NO-cGMP signaling pathway are present in myometrium, relaxation in response to cGMP-elevating agents and analogs occurs at relatively high concentrations compared to vascular smooth muscle. As a result, myometrium has been referred to as refractory or insensitive [12–15].

Pharmacologic manipulation of this pathway has been explored as a means to prevent preterm uterine contractions [7, 16–18] and relieve dysmenorrhea [19–21]. In a rat model however, responses to atrial natriuretic peptide [13] and PKG levels [12] were reduced during gestation, inconsistent with an active role in mediating gestational quiescence. In our previous study, cGMP relaxed myometrial strips in tissues with high PKG (estrogenized rats) but was ineffective in tissues from progesterone-dominated rats having low PKG [12]. The hormonal regulation of both PKG levels and responsiveness to cGMP suggests that this signaling pathway may be important in regulating other myometrial functions involving contractility. The present study was undertaken to test the hypothesis that uterine PKG levels vary with the human menstrual cycle to assist in controlling constriction of the spiral arterioles during menstruation. The results presented here are consistent with a role for PKG in the control of uterine smooth muscle tone contributing to force generation near menstruation and regulation of blood flow to the endometrial lining.

MATERIALS AND METHODS

Source of Myometrial Tissues

Normal human myometrial tissue was obtained from uteri of nonpregnant women undergoing hysterectomy for various gynecologic conditions (Table 1). Informed consent in writing for the use of tissue was obtained from the women undergoing surgery according to protocols approved by the Institutional Review Boards for Human Experimentation at the University of Texas Southwestern Medical Center, the University of Alabama at Birmingham, and Institutional Review Boards of St. Paul (Dallas, TX) and Brookwood (Birmingham, AL) Hospitals. Tissues were dissected free of serosa, connective tissue, and major blood vessels, rinsed in PBS, and snap-frozen in liquid N2 prior to use.


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  		TABLE 1. Clinical characteristics of nonpregnant women from whom uterine specimens were obtained.a

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissues were sectioned at 5 µm and mounted on slides on which both positive (human lung, kidney, adrenal, spleen, and placenta) and negative (human liver, skin) control sections for PKG were mounted [22]. These controls were first utilized to optimize antigen retrieval conditions and antibody dilutions to obtain specific staining for PKG. Then, the slides were used to ensure specificity of results obtained with tissues from the female reproductive tract. After drying in a microwave oven, the slides were deparaffinized in xylene and graded alcohols to water. Epitope retrieval was then performed in a pressure cooker (9 min) using 0.25 M Tris base buffer (pH 9) as the epitope retrieval solution. Endogenous biotin activity was then blocked by placing the slides in dilute egg white solution (one egg white in 100 ml distilled water) for 15 min at room temperature, followed by a distilled water rinse and then incubation in fresh skim milk at room temperature for 15 min [23, 24]. Thereafter, slides were incubated in primary PKG antibody (1:75; StressGen Biotechnologies Corp., Victoria, BC, Canada) for 30 min at 25°C using constant gentle orbital rotation. After a similar incubation with biotinylated secondary antibody (Scytek, Logan, UT) for 15 min, slides were placed in 0.3% H2O2 in PBS for 10–13 min to quench endogenous peroxidase activity. Slides were then incubated with horseradish peroxidase-conjugated streptavidin (Scytek) for 15 min at 25°C. Reaction product was developed by immersing the slides in prepared diaminobenzidine solution (Research Genetics, Huntsville, AL) at 32°C for 4 min. The slides were then rinsed in tap water and placed in 0.5% copper sulfate in normal saline for 5 min at 25°C to enhance the appearance of the chromogen. Finally, the slides were rinsed in water, counterstained in hematoxylin, dehydrated in graded alcohols and xylene, and protected with a coverslip. As a negative control, specimens of the same tissue were stained as described above except the primary antibody was replaced with nonimmune rabbit IgG.

Preparation of Extracts

Frozen tissues were pulverized in liquid N2 and extracts prepared by polytron homogenization (three 10-sec bursts, setting 50%) in three volumes (0.4 ml) of PE buffer (20 mM potassium phosphate, pH 7.0, 150 mM NaCl, 0.32 M sucrose, 2 mM EDTA, 0.1 mM PMSF, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin). Crude soluble and particulate fractions were separated by centrifugation at 12 000 x g for 10 min.

Western Blot Analysis

Proteins in tissue extracts were separated by SDS-PAGE on 10% gels. Proteins were transferred to nitrocellulose at 70 V for 14–16 h in the presence of methanol (20%). Blots were treated with TBST-milk buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1%Tween-20, 4% powdered milk) for 1 h and then incubated overnight at 4°C with a polyclonal PKG peptide antibody diluted (1:5000 or 1:10 000) in TBS containing 2.5% BSA and 0.05% sodium azide (TBS-BSA). Thereafter the blots were washed three times (5 min each) with TBSD (TBS containing 0.05% SDS, 0.05% NP-40, and 0.125% deoxycholate) and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:20 000–1:40 000). After extensive washing with TBST or TBSD, the blots were developed with a chemiluminescent detection system. Relative amounts of PKG were quantified using a Hewlett Packard ScanJet 3c and Scion Image analysis program from Scion Corp. (Frederick, MD). For all Western blots of PKG, the polyclonal antibody used in this study was made by immunizing rabbits against [bu791]a C-terminal peptide of human PKG (DEPPPDDNSGGWDIDF, StressGen Biotechnologies Corp.). This region of the protein is identical in bovine and human species and in both the I{alpha} and Iß isoforms of PKG. This antibody preparation has therefore been used to quantitate total PKG (I{alpha} and Iß) in human extracts by comparison with either standards of purified bovine lung PKG I{alpha} or bovine aortic PKG Iß run on the same blot. In the Western blots presented, PKG I{alpha} (76 kDa) and Iß (78 kDa) comigrate. Uterine tissue expresses predominantly the Iß isoform [25]. For Western blot analysis of smooth muscle myosin II (SMM II), blots were run as for PKG blots except 2 µg extract protein was run per lane (0.5, 1, and 2, but not 5 µg per lane resulted in linear blotting conditions, data not shown). For detection of SMM II immunoreactive protein, blots were incubated in 0.16 µg/ml affinity-purified rabbit polyclonal antibody to SMM II diluted in TBS-BSA [26]. For secondary antibody, goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad Laboratories, Richmond, CA) was used at a dilution of 1:10 000. Immunoreactive bands were visualized by incubating blots with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidinium (Gibco BRL Life Technologies, Inc., Gaithersburg, MD).

Assay for PKG

Activity of PKG was determined from crude soluble extracts. Incorporation of 32P from [{gamma}32P]ATP into BPDE-tide was monitored using a filter paper assay as described previously [12, 14]. Extracts were prepared in PE buffer containing 1% Triton X-100 in order to extract maximal cGMP-stimulated activity. Most (90%) of the immunoreactive PKG was recovered without Triton X-100 extraction, although the presence of the detergent may increase cGMP-stimulated PKG activity. Decreased levels of cGMP-stimulated activity may be obtained in the presence of other enzymes competing for ATP, phosphodiesterases degrading cGMP, or the presence of undefined inhibitory substances in crude extracts. Assay conditions were optimized for recovery and measurement of active enzyme, and tissues were analyzed together in the same assay.

Statistical Analysis

Results are expressed as means ± SEM. Statistical comparisons between multiple groups were conducted with a one-way ANOVA followed by posthoc Student Neuman Keuls test. A P value of <0.05 was considered significant.

RESULTS

Immunohistochemistry was utilized to determine the distribution of PKG expression in reproductive tissues from nonpregnant women (Figs. 1 and 2). In myometrial tissues obtained from the proliferative phase, immunoreactive staining was localized to uterine and vascular smooth muscle cells. Immunoreactivity was more intense in the arterial smooth muscle cells compared with uterine myocytes (Fig. 1A). Whereas PKG immunoreactivity was consistently absent in endothelial cells of arterioles, positive staining for PKG was observed in some, but not all, venous endothelial cells (not shown). Expression of PKG was absent in stromal cells surrounding the myometrial muscle bundles and vascular plexus (Fig. 1A).



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  		FIG. 1. Immunohistochemical localization of PKG in human uterine tissues. Immunostaining was performed with a polyclonal antibody against a C-terminal peptide of human PKG. Negative controls (insets in A and C) were obtained with nonimmune rabbit IgG substituted for the primary antibody. Hematoxylin was used for counterstaining. A) Myometrial tissue from proliferative phase. Immunoreactivity is visualized in myometrial and vascular smooth muscle cells. Stromal cells separating the myocyte bundles are negative. B) Cervical tissue obtained during the proliferative phase of the menstrual cycle. The PKG staining is observed in vascular smooth muscle and a small percentage of cervical stromal cells (arrowheads). C) Proliferative endometrium. Immunostaining is visualized in a subset of endometrial stromal cells underlying the basement membrane of the glandular epithelium (arrows). Epithelial cells and the majority of endometrial stromal cells are negative for PKG. D) Magnification of C demonstrating a subset of PKG-positive cells adjacent to endometrial glands (arrows). Panels A–C are x100 magnification and D is magnified x200. Replacement of nonimmune IgG for primary antibody resulted in negative staining in most cells except for weak, light brown staining in epithelial cells visible at higher power

In cervical tissues, PKG expression was confined to vascular smooth muscle and a small percentage of cervical stromal cells (Fig. 1B). In proliferative endometrium (Fig. 1, C and D), glandular epithelial cells of the endometrium and the majority of endometrial stromal cells were not immunoreactive for PKG. However, stromal cells underlying the basement membrane of glandular epithelium stained positive for PKG.

Distribution of PKG immunoreactivity in uterine tissues from the secretory phase is depicted in Figure 2. Like proliferative phase myometrium, vascular smooth muscle showed intense staining for PKG. Staining was very weak in myometrial smooth muscle cells, however, compared with vascular smooth muscle cells in the same section (Fig. 2A). The striking differences in staining intensity between vascular smooth muscle and myometrial cells were observed in three different uteri from the secretory phase and in all sections examined. In secretory endometrium, greater numbers of endometrial stromal cells were positive for PKG. In uterine and vascular smooth muscle cells, distribution of PKG was mainly cytoplasmic and perinuclear. The subcellular distribution of staining in the smaller cervical cells (Fig. 1B) and endometrial stromal cells (Fig. 1C) is more difficult to determine by this technique, and a nuclear distribution cannot be ruled out. The majority of endometrial stromal cells separating the nonreactive secretory endometrial glands were immunoreactive for PKG (Fig. 2B).



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  		FIG. 2. Immunohistochemical localization of PKG in secretory phase uterine tissues. A) Secretory myometrium. The PKG is localized to vascular smooth muscle. Staining intensity in myometrial smooth muscle bundles was decreased substantially relative to that of the adjacent vasculature. B) Secretory endometrium. The PKG was localized to the majority of endometrial stromal cells separating the secretory glands. Only faint nonspecific staining in glandular epithelial cells was observed in serial sections of identical tissues incubated in all reagents except nonimmune rabbit IgG substituted for the primary antibody (not shown). e, Endometrium; m, myometrium. Original magnification x100

To quantify PKG in myometrium from different menstrual cycle phases, Western blot analysis was performed. The clinical characteristics of women from whom specimens were obtained are detailed in Table 1. The intent was to obtain samples during various phases of the menstrual cycle and after menopause. To ensure that Western blot analysis would allow detection of differences in myometrial PKG from uterine specimens taken from individuals in different menstrual cycle phases, crude soluble uterine extracts prepared from two specimens (C and F from Table 1) were analyzed along with standards of purified bovine aorta and lung PKG. Different amounts of extract protein (5–20 µg per lane) and pure PKG (2–50 ng) were applied to the gels. Western blot analysis (Fig. 3A) revealed a single immunoreactive band comigrating with the purified PKG standards. The intensities of immunoreactive bands were linear with respect to amount of extract protein between 5 and 20 µg for specimen C. For specimen F, the arbitrary units of the PKG bands were not linear beyond 10 µg extract protein. For quantification of all specimens, 10 µg crude soluble extract was applied per lane.



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  		FIG. 3. Western blot analysis and quantification of PKG in uterine extracts. A) Levels of PKG in myometrial extracts from two women (designated C and F) were examined by Western blot analysis. Standards of purified PKG I{alpha} and Iß were included. B) Immunoreactive PKG was quantified by scanning densitometry and the data expressed as arbitrary units as a function of purified PKG (ng) or extract protein (µg). Linear conditions for PKG quantification were established between 5 and 20 µg extract protein for specimen C. For specimen F, the arbitrary units of the PKG bands were not linear between 10 and 20 µg extract protein

A Western blot of myometrial tissues from 19 nonpregnant women is presented in Figure 4. Of the 19 cases analyzed, only 1 could not be clearly identified with respect to menstrual cycle phase (specimen N), and this sample was not included in subsequent analyses. In myometrial extracts, a single immunoreactive band comigrating with the purified standard is visible. The PKG bands were quantitated by densitometry, and the samples were grouped according to menstrual cycle phase (Fig. 5). The data from myometrial specimens from women with endometrial histology indicative of proliferative phase but exhibiting stromal progesterone changes and history of progestin treatment were combined with data from myometrial specimens exhibiting characteristics of endogenous progesterone exposure (secretory phase). Myometrial samples obtained from proliferative phase premenopausal women had the highest PKG levels of all specimens analyzed. Myometrial specimens from tissues characterized as secretory or secretory with menstrual changes consistently fell at the lower end of the spectrum of levels of PKG. The expression of PKG was significantly increased in myometrium from proliferative phase premenopausal or postmenopausal women relative to that in secretory phase myometrium (P < 0.01). The PKG in secretory phase myometrium was less than proliferative phase or postmenopausal groups (P < 0.01) whether or not cases with exogenous progestin administration were included. Values for proliferative phase and postmenopausal myometrial PKG were not significantly different.



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  		FIG. 4. Western blot analysis for PKG in human myometrial extracts in different phases of the menstrual cycle. Using conditions determined to allow quantification of PKG (see Fig. 3), blotting was carried out on protein extracts (10 µg) of myometrial tissues from 19 different women. Letter codes were assigned to each specimen and described in Table 1



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  		FIG. 5. Relative levels of PKG as a function of menstrual cycle phase. Scanning densitometry was used to quantify the relative expression of PKG in Western blots (as shown in Fig. 4). Specimens were grouped as proliferative (open bar), secretory (myometrium from the luteal phase or with progestin administration, solid bar), and postmenopausal (hatched bar). Expression of PKG is decreased significantly in myometrium from secretory phase compared with that from proliferative phase (P < 0.01) or from postmenopausal women (P < 0.01)

Activity assays for PKG were conducted on freshly prepared myometrial extracts using a selective substrate and the data were expressed as a function of the level of immunoreactive protein (Fig. 6). Eight of the 19 myometrial specimens that contained a range of PKG levels (including the lowest and highest) as determined by Western blot analysis were chosen for enzyme activity assays. Cyclic GMP-stimulated activity was significantly decreased (P < 0.03) in myometrial samples obtained from women exposed to progesterone or progestin (88.7 ± 7.1, mean ± SEM for specimens C, J, and H, Table 1) compared with those from women not exposed to progesterone (150.6 ± 8.6, mean ± SEM for specimens S, P, D, B, and F listed in Table 1). The levels of immunoreactive PKG in uterine extracts correlated with the levels of enzymatic activity (r = 0.84).



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  		FIG. 6. Correlation of PKG levels with enzymatic activity. Eight uterine smooth muscle specimens (described in Table 1) were further analyzed for cGMP-stimulated protein kinase activity. The values plotted represent cGMP-stimulated pmol 32P/min·mg protein incorporated into BPDEtide. Cyclic GMP-stimulated activity was plotted as a function of the levels of immunoreactive PKG (Fig. 4 and Table 1). Samples from proliferative phase or menopause (open circles) were identified as S, P, D, B, and F in Table 1. Progestin-exposed tissues (solid circles) were identified as C, J, and H in Table 1 (listed in order of increasing PKG levels).

It was also important to compare PKG levels relative to those of a protein that does not change with the menstrual cycle. For this purpose, SMM II was analyzed [26], and levels of PKG relative to SMM II were computed (Fig. 7). Changes in PKG levels during the menstrual cycle were distinct from the monophasic expression of smooth muscle myosin. These data indicate that although myometrial amounts of the major smooth muscle contractile protein (myosin, µg/mg protein) do not change during the menstrual cycle, PKG is down regulated during the progesterone-dominated phase.



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  		FIG. 7. Levels of PKG and SMM II in human myometrial extracts. The PKG levels were compared with levels of SMM II in extracts from eight women. Codes for tissues (x axis) are described in Table 1. The inset illustrates a Western blot for SMM II. Levels of SMM II were determined by densitometric analysis and compared to levels of PKG

DISCUSSION

Uterine smooth muscle contractility contributes to a number of reproductive functions. An increased force of contraction by uterine smooth muscle cells together with vascular changes can obstruct endometrial blood flow and lead to tissue disintegration. Fundo-cervical uterine smooth muscle contractions can then contribute to the shedding of the endometrial lining. In addition, expulsion of the fetus at the end of gestation is brought about by intense contractions of interdigitating smooth muscle bundles [3]. It is therefore likely that uterine smooth muscle cells may have different regulatory mechanisms for contraction and relaxation depending on the particular uterine function, the smooth muscle architecture, and the hormonal milieu. We have focused on the NO-cGMP signaling pathway, as it has been identified as mediating vascular smooth muscle relaxation [5, 6, 8]. The current study was undertaken to test the hypothesis that uterine PKG levels may be subject to regulation by the fluctuations in ovarian hormones during the human menstrual cycle.

Although high levels of PKG can be extracted from uterine tissues, little is known about the localization of the enzyme within the uterus. Our studies confirm that there is abundant myometrial PKG in proliferative phase myocytes. Staining intensity in myocytes from the secretory phase was substantially lower than that of vascular smooth muscle. This result was confirmed by quantification by Western blot analysis. Unlike myometrial smooth muscle PKG, kinase levels present in myometrial blood vessels do not appear to decrease in the secretory phase. Thus, it is likely that our estimates of secretory phase uterine smooth muscle PKG levels are overestimated due to the unavoidable contamination of vascular smooth muscle in myometrial tissue homogenates.

To our knowledge, this is the first report of PKG in endometrial stromal cells. By immunohistochemistry, endometrial stromal cells acquired expression of PKG during the secretory phase suggesting an important, but yet unknown, function for PKG in these cells. NO has been implicated as an important mediator of endometrial function [27]. These results indicate that PKG may mediate at least some effects of NO in this cell type. The presence of PKG in stromal cervical cells also suggests that this kinase may mediate effects of NO in the cervix [28].

Previously, we reported that in rat myometrium, PKG levels were regulated by ovarian hormones [12]. The data reported herein are consistent with an upregulation of human myometrial PKG protein during the proliferative phase, when estradiol levels are highest. Interestingly, postmenopausal specimens had similar PKG levels. The maintenance of PKG levels in the postmenopausal period may be attributable to the absence of progesterone and decreased levels of estrogen-stimulated nuclear progesterone receptors, rather than any estrogenic stimulation. This interpretation is consistent with our finding that PKG levels are significantly decreased in myometrial tissues during the secretory phase, suggesting suppression of PKG expression by progesterone. In gestational rat myometrium, PKG levels remained low through term [12], even though estradiol levels increase while progesterone levels decrease. Although it is possible that the levels of estrogen in the rat at term are not high enough to induce PKG expression (as in estrogen administration to ovariectomized animals), in sum the data are more consistent with PKG down regulation by progesterone, even the relatively low levels present at term. Furthermore, uteri from individuals receiving exogenous progestins (e.g., Depo-Provera) also contained lower levels of PKG.

An interesting observation is that PKG is regulated differently within the distinct uterine compartments. In myometrial smooth muscle, progestins lead to a down regulation of PKG, while endometrial stromal cell PKG may increase. The placenta is an abundant source of PKG [29], and therefore PKG expression is also maintained in this tissue despite exposure to high concentrations of progestins. It is clear that PKG expression is subject to hormonal regulation and that complex mechanisms regulate PKG expression within the reproductive tract.

The physiologic roles of NO and the downstream signals mediating NO effects are pleiotrophic. In uterine smooth muscle, NO has been shown to produce both inhibitory and excitatory contractile responses [30–32]. The cGMP-dependent component of NO action is thought to mediate relaxation, while the cGMP-independent action of NO may increase contractility by activation of prostaglandin synthesis [30]. Other cGMP-independent pathways for NO-mediated relaxation may involve direct activation of smooth muscle potassium channels resulting in hyperpolarization [33, 34]. An issue that remains to be clarified is understanding which conditions promote NO-dependent contraction and which conditions are conducive to cGMP-dependent or -independent relaxation. By identifying regulation of PKG levels with the menstrual cycle, we provide information on when and where cGMP- and PKG-mediated effects of NO might be expected to occur.

Physiological substrates for PKG have been identified, most of which play a role in mediating smooth muscle relaxation (e.g., phospholamban [9], inositol triphosphate receptor [10], K+ channel subunits [11], myosin-binding subunit of myosin phosphatase [35]). Many other potentially physiological substrates have been identified; however, their functions in mediating PKG signaling are not yet as well defined. Recent work has identified PKG as a mediator of gene transcription. Activation of PKG has been associated with induction of c-fos mRNA expression [36, 37]. Thus, it is possible that PKG may play a role in orchestrating gene expression in uterine tissues throughout the menstrual cycle. The essential role of PKG in mediating smooth muscle relaxation does not exclude other functions for the kinase that may regulate complex processes such as menstruation, implantation, and parturition. Further studies are underway to define the physiologic significance of endometrial PKG and hormonal regulation of PKG expression during the luteal phase and pregnancy.

ACKNOWLEDGMENTS

The authors thank Cheryl Van Epps-Fung, M.S. for technical assistance. We acknowledge the contributions of Drs. Edward Wilson, Michael Connor, and the members of the Pathology Departments of UAB, St. Paul, and Brookwood Hospitals. We also thank the UAB Comprehensive Cancer Center Tissue Procurement Facility and personnel for assistance in procuring tissues. Thanks to Dr. Sharron Francis for providing purified PKG Iß. Richardson's Meat Processing and Greene's Processing Centers are acknowledged for donating bovine lungs for the purification of PKG.

FOOTNOTES

First decision: 22 May 2000.

1 This study was supported by grants NIH HD32622 (T.L.C.), NIH P01-HD11149 (R.A.W.), and NIH HL59618 (P.deL.). Back

2 Correspondence: Trudy L. Cornwell, Department of Pathology, VH Room G019, University of Alabama at Birmingham, 1670 University Blvd., Birmingham, AL 35294-0019. FAX: 205 934 1775; cornwell{at}path.uab.edu Back

Accepted: October 27, 2000.

Received: April 17, 2000.

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