Nitric oxide relaxes human myometrium by a cGMP-independent mechanism

Karri K. Bradley1, Iain L. O. Buxton1, James E. Barber2, Terrence McGaw2, and Michael E. Bradley1

Departments of 1 Pharmacology and 2 Obstetrics and Gynecology, University of Nevada, Reno, Nevada 89557

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The role of intracellular guanosine 3',5'-cyclic monophosphate concentration ([cGMP]i) in nitric oxide (NO)-mediated relaxations in the uterus has become controversial. We found the NO donor S-nitroso-L-cysteine (CysNO) to potently (IC50 = 30 nM) inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased [cGMP]i significantly (P < 0.001), and this increase was blocked by the guanylyl cyclase inhibitors methylene blue (10 µM) or LY-83583 (1 µM); however, pretreatment with these guanylyl cyclase inhibitors failed to block CysNO-mediated relaxations. Intracellular cAMP concentrations were not altered by treatment of tissues with 10 µM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or beta -phenyl-1,N2-etheno-cGMP did not significantly affect spontaneous contractility. Pretreatment of tissues with charybdotoxin [a calcium-dependent potassium channel (BK) blocker] completely reversed CysNO-induced relaxations. We conclude that NO is a potent inhibitor of spontaneous contractile activity in the nonpregnant human uterus and that, although guanylyl cyclase and BK activities are increased by NO, increases in [cGMP]i are not required for NO-induced relaxations in this tissue.

guanosine 3',5'-cyclic monophosphate; contraction

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

THE SIGNALS AND MECHANISMS responsible for the control of uterine contractility remain poorly understood. Although agents such as oxytocin, prostaglandins, and catecholamines can clearly alter myometrial tension when administered exogenously, a clearly defined, significant role for any endogenous factor in the onset of contractions (either those associated with normal labor or those that occur during preterm labor) in the human uterus has yet to be shown. The possibility that uterine contractions result not from the appearance of contracting factor(s) but rather from the disappearance of relaxing factor(s) has prompted a number of studies that have assessed the role of the smooth muscle-relaxing factor nitric oxide (NO) in the regulation of uterine contractility. Effects of exogenous NO have now been demonstrated in uterine preparations from a number of species, including rat (5, 7, 9, 21), guinea pig (19), sheep (8), monkey (20), and human (4, 33). These findings suggest that NO donors are in fact capable of altering uterine contractility, either by stimulating contractions (9) or by inhibiting spontaneous [4, 5, 33-35; see also the review by Sladek et al. (29)] or agonist- or KCl-evoked (7, 19, 21, 29) contractions. The mechanism by which NO is believed to alter contractility in the uterus has, however, become rather controversial. In a number of instances, NO effects on uterine tension have been either assumed or stated to be mediated by changes in intracellular cGMP concentrations ([cGMP]i; see Refs. 4, 15, 34), and it has been proposed that a NO right-arrow guanylyl cyclase right-arrow cGMP pathway may account for the quiescence of the gravid uterus before the initiation of labor (4, 34). Other studies, however, have been unable to demonstrate a correlation between changing cGMP concentrations in myometria from either rodents (5, 6, 14, 19) or nonpregnant primates (20, 33), bringing into question the role of cGMP in the regulation of uterine contractility. The current studies were designed to address the role of [cGMP]i in NO-mediated relaxations in the nonpregnant human myometrium by examining 1) the effect of guanylyl cyclase inhibition on NO-induced relaxations, 2) the effect of NO on [cGMP]i, both in the presence and absence of guanylyl cyclase inhibitors, 3) the ability of cGMP analogs to alter uterine contractility, and 4) the role of large-conductance calcium-dependent potassium channels (BK) in NO-mediated relaxations. We find that, while activation of BK is involved in NO-induced relaxations, increased [cGMP]i do not appear to be required for NO-induced relaxations in the nonpregnant human myometrium.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Preparation and treatment of uterine samples. Experimental methods were essentially as described previously (20). Contractile studies were performed on samples of nonpregnant human uterine tissue obtained from midcycle, informed, and consenting women undergoing hysterectomy for elective sterilization or for uterine pathologies that did not involve the myometrial parenchyma; records were kept and statistically analyzed to ensure that variables such as age, race, parity, type of anesthesia, drug use history, etc., had no effect on experimental outcomes. Tissue samples were transported to the laboratory in a cold physiological buffer containing (in mM) 120 NaCl, 5 KCl, 0.587 KH2PO4, 0.589 Na2HPO4, 2.5 MgCl2, 20 alpha ,D-glucose, 2.5 CaCl2, 25 Tris, and 5 NaHCO3; this buffer was also used during the course of organ bath experiments. Strips of myometrium were carefully cut from the center of the tissue section, mounted in organ baths (Radnoti, Monrovia, CA), and attached to isometric force transducers (Kent Scientific, Litchfield, CT) by silk thread. Transducer voltages were amplified and converted to digital signals by an analog-to-digital board mounted within a computer system running the Workbench data acquisition system (Strawberry Tree, Sunnyvale, CA). Strips were maintained at 37°C, aerated with 100% O2, and loaded with initial tensions of 1.0 g/mm2; initial tensions were based on empirical results obtained during pilot experiments that established force-length relationships in nonpregnant human myometrium and correspond to tissue lengths that are 1.5× the resting tissue length. During the course of a 1-h equilibration period, tissues were repeatedly challenged with contractile agonists such as oxytocin to ensure contractile viability, followed by bath washout and relaxation periods.

NO was added to tissues in the form of S-nitroso-L-cysteine (CysNO) from a concentrated stock solution for which water served as the solvent. CysNO was prepared by the method described by Gibson et al. (11). The control treatment for the NO donor was "spent" CysNO (i.e., CysNO that had been bubbled with oxygen for 30 min). Concentration-response curves for CysNO were constructed in a noncumulative manner, as were those constructed for the cGMP analogs 8-bromo-cGMP (8-BrcGMP) and beta -phenyl-1,N2-etheno-cGMP (PET-cGMP).

Simultaneous measurement of tissue tensions and intracellular cyclic nucleotide concentrations. Spontaneously active tissue strips mounted within organ baths were treated with CysNO and flash-frozen in liquid nitrogen precisely 30 s after CysNO addition; basal samples were frozen at a point midway between spontaneous contractions. Without allowing samples to thaw, tissues were homogenized with a Duall glass-glass homogenizer in 1 ml of 6% trichloroacetic acid dissolved in acetone while constantly immersed in a methanol-dry ice slurry. Precipitated protein was removed by centrifugation, acid was removed by triplicate extraction with diethyl ether, and residual ether was evaporated by heating samples to 70°C for 10 min. After lyophilization, nucleotide samples were resuspended in phosphate buffer and assayed for either cGMP or cAMP by enzyme-linked immunoassay using antibodies obtained from Cayman Chemical (Ann Arbor, MI). Protein present in the acid precipitate was neutralized with NaOH and assayed by the method of Bradford (3).

To inhibit guanylyl cyclase activities, tissue strips mounted in organ baths were pretreated with methylene blue (10 µM) or LY-83583 (1 µM) for 30 min at 37°C in the dark before the addition of either CysNO or ion channel blockers. Guanylyl cyclase treatments were considered to be irreversible; concentrations of methylene blue and LY-83583 used were determined empirically as sufficient to completely inhibit guanylyl cyclase but to be incapable of altering spontaneous contractility. Preliminary experiments were performed to determine time courses of cGMP or cAMP generation in response to treatment with CysNO, or after pretreatment with cyclase inhibitors.

Data analysis. Spontaneous contractile activity was quantified by integration of the area under each contractile record using software written specifically for this purpose. To evaluate the effects of additions, spontaneous activity was quantified for periods of 5 min before and after CysNO addition or for 15 min before and after cGMP analog addition; effects were then expressed as a percentage of the amount of activity present immediately before addition to control for any differences in spontaneous activity during the course of an experiment and to control for any cumulative effects of treatments. Values for contraction or cyclic nucleotide concentrations were evaluated by one-way analysis of variance; results presented are statistical means ± SE, and n refers to the number of patients contributing a minimum of four tissue strip samples to each experimental set. Assays for cyclic nucleotide concentrations were performed in duplicate for each tissue strip. Curve fitting and analyses were performed with the aid of Graphpad Prism graphics software (Carlsbad, CA).

Materials. All compounds were made up in water except for LY-83583 (RBI, Natick, MA), which was dissolved in 0.45% ethanol; under no circumstances was alcohol present in organ baths at a final concentration exceeding 0.003%. PET-cGMP was obtained from Ruth Langhorst Biolog International (La Jolla, CA), 8-BrcGMP was from RBI (recently acquired by Sigma Chemical, St. Louis, MO), SQ-22536 was from LC Laboratories (Woburn, MA), and cyclic nucleotide enzyme-linked immunoassay kits were obtained from Cayman Chemical. All other compounds were reagent grade and were obtained from Sigma Chemical.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Addition of the NO donor CysNO to organ baths containing spontaneously active tissue strips from nonpregnant human myometrium resulted in an immediate and profound inhibition of contractile activity (Fig. 1). This effect was highly reproducible and present in every tissue sample assayed; the effect was also transient, as spontaneous activity resumed on either bath washout or a period of 5-8 min after addition of NO donor. Similar results were obtained when native (gaseous) NO was used (not shown). Addition of CysNO that had been reacted with 100% O2 for 30 min (spent CysNO) was never found to affect contractility (Fig. 1, inset); bath pH was routinely monitored but did not change from pH 7.40 with addition of any of the reagents used in the study.


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Fig. 1.   Nitric oxide inhibition of spontaneous uterine contractility. S-nitroso-L-cysteine (CysNO) was added to spontaneously active human myometrium at the arrow and was present for the duration of the recording. Failure of the control (spent CysNO) to alter spontaneous contractility is illustrated in the inset. Results are representative of 30 patients.

The relaxing effects of CysNO were quantified by means of integration of the area under the contractile record, permitting the establishment of a concentration-response relationship between CysNO and tissue tension. Increasing concentrations of CysNO resulted in greater inhibition of spontaneous activity; the IC50 for this effect was calculated to be ~30 nM (Fig. 2). Possible cumulative effects of CysNO treatment, as well as the effects of decreasing spontaneous activities throughout, were ruled out by expressing the quantitative data as a percentage of the spontaneous activity immediately preceding each experimental treatment.


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Fig. 2.   CysNO concentration ([CysNO])-response relationship. Increasing concentrations of CysNO were added in a noncumulative manner to strips of spontaneously contractile human myometrium; results were quantified by integration under the contractile record as described in EXPERIMENTAL PROCEDURES. Results are means ± SE and were obtained from experiments performed on tissue samples from 9 women. IC50 for CysNO was calculated from the curve fit to be 30 nM.

[cGMP]i were determined at precise time points during the course of either spontaneous contractions or CysNO-induced relaxations to establish the temporal relationship between [cGMP]i and tissue tension. Addition of CysNO to spontaneously active tissue strips resulted in a significant fivefold increase in [cGMP]i (Fig. 3A) when compared with basal levels. Pretreatment of tissues with the guanylyl cyclase inhibitors methylene blue (10 µM) or LY-83583 (1 µM) completely blocked the CysNO-induced increase in [cGMP]i (Fig. 3A). Simultaneous recording of tissue tensions revealed a statistically significant 80% reduction in spontaneous contractility upon CysNO treatment (Fig. 3B); pretreatment with either guanylyl cyclase inhibitor had no effect, however, on the ability of CysNO to relax the tissue. CysNO therefore appeared to be capable of blocking spontaneous contractile activity even in the demonstrable absence of guanylyl cyclase activation. Guanylyl cyclase inhibitors themselves had no effect on the level of spontaneous activity (e.g., see Fig. 6).


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Fig. 3.   CysNO relaxation of myometrium in the absence of increases in intracellular cGMP concentration ([cGMP]i). A: [cGMP]i were determined in strips of myometrium that were relaxed (basal), treated with 10 µM CysNO alone, or treated with CysNO after a 30-min pretreatment with either 10 µM methylene blue (MB) or 1 µM LY-83583. Results presented are means ± SE and are derived from duplicate assays on strips obtained from 4 patients. Mean indicated by dagger  was found to be statistically different (P < 0.001) from the means indicated by *, which were not significantly different from one another. B: quantification of spontaneous contractile activity at rest (basal) or after treatment with 10 µM CysNO in myometrial tissue strips either untreated (CysNO) or pretreated for 30 min with methylene blue (10 µM) or LY-83583 (1 µM). Results are expressed as a percentage of the spontaneous activity seen immediately before each treatment and are means ± SE from the same samples used in A; n = 4 patients. Mean denoted by dagger  is significantly different from the means denoted by * (P < 0.001), which are not significantly different from one another.

To examine whether cGMP-dependent second messenger activities were involved in the CysNO-induced relaxations, tissues were incubated with the cGMP analog 8-BrcGMP or PET-cGMP; these compounds are both relatively more stable to hydrolysis and permeant than native cGMP, and the latter compound is thought to be selective for the predominant cGMP-dependent protein kinase (PKG) isoform (1beta ) found in the uterus (26, 28). Although tissues were treated for 30 min with concentrations of analog as high as 1 mM, no statistically significant effect on tissue tension was seen in response to either compound (Fig. 4), suggesting that activation of distal intracellular messengers such as PKG is also not required for CysNO-induced relaxation of human myometrium.


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Fig. 4.   cGMP analogs have no effect on uterine contractility. Myometrial tissue strips were incubated with increasing (noncumulative) concentrations of the cGMP analogs 8-bromo-cGMP ([8-BrcGMP]) or beta -phenyl-1,N2-etheno-cGMP ([PET-cGMP]) for 30 min. Data points are means obtained from 4 women ± SE; linear regression slopes were not significantly different from 0.

Because cross talk between adenylyl and guanylyl cyclase systems is known to exist (18), we examined the possibility that CysNO signals uterine relaxations via increases in intracellular cAMP concentration ([cAMP]i). Treatment of tissue strips with 10 µM CysNO failed to result in an increase in [cAMP]i (Fig. 5); that an intact adenylyl cyclase-cAMP system was present in these tissues was demonstrated by the ability of isoproterenol to stimulate increases in [cAMP]i and the ability of the adenylyl cyclase inhibitor SQ-22536 to inhibit these increases (Fig. 5). Additional evidence for an intact adenylyl cyclase system in these tissues was the observation that SQ-22536 completely reversed the ability of isoproterenol to relax these tissues (not shown); these findings in the adenylyl cyclase/cAMP system serve as an internal control for the ability of cyclic nucleotides to affect tissue tensions and provide an excellent point of contrast with the results we obtained using inhibitors of guanylyl cyclase.


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Fig. 5.   Increases in intracellular cAMP concentration ([cAMP]i) are not involved in CysNO-mediated relaxations. [cAMP]i was determined in myometrial tissue strips that were at rest (basal), treated with 10 µM CysNO alone, or treated with 10 µM CysNO after 30 min pretreatment with the adenylyl cyclase inhibitor SQ-22536 (30 µM). As a positive control, tissues were treated with isoproterenol (30 µM) to stimulate increases in [cAMP]i; these increases were blocked by pretreatment with SQ-22536. Results are means ± SE from duplicate assays on tissues from 4 women; * statistically significant difference compared with the basal mean (P < 0.05).

To determine whether BK might be involved in CysNO-induced relaxations in human myometrium, tissues were pretreated with charybdotoxin, a relatively specific BK blocker. Charybdotoxin pretreatment reversed CysNO inhibition of spontaneous contractility in a statistically significant manner (Fig. 6); similar results were also obtained when tissues were pretreated with either tetraethylammonium chloride (1 mM) or iberiotoxin (100 nM; not shown). Methylene blue was present in all tissue samples to exclude any cGMP-dependent effects of BK blocker, although identical results were obtained (although not shown) in the absence of methylene blue, which argues against a cGMP-dependent effect of charybdotoxin on NO-induced relaxations.


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Fig. 6.   CysNO effects are blocked by charybdotoxin. Strips of myometrium were incubated in either methylene blue (MB, 10 µM) alone, methylene blue plus CysNO (10 µM), or methylene blue plus CysNO after pretreatment of the tissues with 100 nM charybdotoxin (ChTx) for 15 min. Results are means ± SE from replicate tissues obtained from 4 women; dagger  denotes mean value, which is significantly (P < 0.001) different from the means denoted by *, which are not statistically different from one another.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The majority of reports describing the effects of NO or NO donors on uterine contractility have been based on results obtained from rodent models (9, 14, 15, 19, 34, 35). That exogenous NO donors can affect uterine contractility is now generally accepted; what remains unclear is whether guanylyl cyclase activation is required for these effects, as is clearly the case in NO-mediated relaxation in many nonuterine smooth muscles (see Ref. 22 for review). The main goal of the current study was therefore to determine whether changes in [cGMP]i are required for NO-induced relaxation in the human myometrium; in the course of our work, we also explored a possible component of the cGMP-independent mechanism of NO action in this tissue.

Quantitative analysis of contractile records from 30 women revealed a highly significant inhibitory effect of CysNO on spontaneous myometrial contractility (Figs. 1, 2, and 3B). CysNO appears to be a much more potent inhibitor (IC50 = 30 nM) of uterine contractility than either sodium nitroprusside or diethylamine-NO, which must be used at much higher concentrations to achieve maximal effects in rats and humans, respectively (4, 35); it is possible and even likely, however, that these differences in potency are due to the reproductive status of the uterine tissue studied. In our experiments, addition of spent CysNO to organ baths did not evoke a tissue response, bath pH did not change after any addition in the study, effects of CysNO were mimicked by treating tissues with native NO, and tissue viability was not compromised due to CysNO treatments. These findings support the contention that the effects of CysNO addition to organ baths were due to specific liberation of NO and were not the result of changing bath pH, tissue damage, or nonspecific effects of the donor.

In agreement with earlier findings of others in rodents (5, 6, 14) and humans (33), careful quantitation of intracellular cyclic nucleotide concentrations in tissue strips that were actively contracting and relaxing revealed significant increases in [cGMP]i in myometria from nonpregnant women (Fig. 3A). However, pretreatment of tissues with inhibitors demonstrably capable of blocking the CysNO-induced rise in [cGMP]i (Fig. 3A) had no effect on the ability of CysNO to block spontaneous contractions in these same samples (Fig. 3B). These findings suggest that, even though guanylyl cyclase serves as a target for CysNO in nonpregnant human myometrium, activation of this enzymatic activity is not required for CysNO-induced inhibition of spontaneous contractility. That cGMP is not involved in CysNO-mediated relaxations in the uterus is also supported by our inability to demonstrate a significant effect of cGMP analogs on uterine contractility (Fig. 4), despite 30-min incubations with high (<= 1 mM) concentrations of analog. These latter findings are consistent with earlier results obtained from both rat (5, 14) and human (33) myometrium and also indirectly support recent findings by Hennan and Diamond (14) that suggest that PKG activation is not correlated with NO-induced relaxations in the rat uterus.

Our inability to demonstrate a role for [cGMP]i in NO-induced relaxations in the uterus is consistent with the conclusions of Diamond and Hennan (5, 14) and with work performed by Word et al. (33) in the nonpregnant human myometrium; our conclusions contrast, however, with those of Garfield and co-workers, who have proposed that NO-stimulated increases in [cGMP]i are involved in the reduction in uterine tissue tensions observed in response to NO donors in both rodents (15, 16, 34, 35) and humans (4, 16). One possible explanation for these discrepancies is that conclusions from previous studies that attributed a role to [cGMP]i in NO-induced uterine relaxation were based on a correlative rather than causal relationship between changes in [cGMP]i and tissue tension. Because NO donors are known to generate distinct forms of NO (29), it is also possible that different NO donors can affect different components of the intracellular signaling pathways within uterine myocytes (e.g., CysNO could be producing relaxations via interaction with a component of the transduction cascade distal to both cGMP and PKG). Compartmentalization of enzymes involved in cGMP-mediated intracellular signaling is also a possible result of treatment of tissues with guanylyl cyclase inhibitors, although the fact that we are able to clearly detect large increases in [cGMP]i would tend to argue against this mechanism as the reason for the failure of guanylyl cyclase inhibitors to reverse CysNO-induced relaxations in these tissues. Resolution of a number of questions relating to the involvement of intracellular cGMP in uterine contraction/relaxation would be facilitated by the consistent use of available and ever-improving inhibitors of guanylyl cyclase whenever these questions are addressed experimentally.

There are a growing number of reports of cGMP-independent effects of NO in a variety of tissue types (1, 2, 10, 12, 13, 17, 23-25, 27, 30-32, 36). In smooth muscle specifically, Hata and co-workers (23, 30, 32) have now clearly demonstrated a cGMP-independent effect of NO in the rat colon. As yet, the mechanism by which NO exerts cGMP-independent effects has not been established, although several possible mechanisms have been suggested. In the vasculature, for example, it has been suggested that NO relaxes smooth muscle by inhibiting a cytochrome P-450 activity that would otherwise act to inhibit BK, ultimately leading to vascular relaxation (1, 13, 36). Alternatively, it has been suggested that NO might interact directly with BK in vascular smooth muscle, resulting in hyperpolarization and relaxation (2). In the present study, we find that blockade of BK by charybdotoxin blocks the ability of NO to inhibit spontaneous contractility in human myometrium; although the involvement of [cGMP]i and PKG in NO effects is unlikely given our results, we have no evidence that NO acts in a direct manner to relax myometrium. Our finding that charybdotoxin blocks the relaxing effects of NO differs from that obtained by Hennan and Diamond (14), who found no effect of charybdotoxin on the relaxing effects of NO (donated by S-nitroso-N-acetylpenicillamine) in the nonpregnant rat myometrium. Although it is (again) possible that these disparate findings are due to differences in respective choices of NO donor, it seems more likely that species differences in myometrial responsiveness to NO are responsible, particularly given our earlier finding that CysNO failed to alter spontaneous (but not agonist-evoked) contractions in the guinea pig uterus (19); these studies also demonstrated the cGMP-independent relaxing effect of CysNO on agonist-evoked contractions in both pregnant and nonpregnant guinea pig myometrium.

In conclusion, we find the NO donor CysNO to be a potent inhibitor of spontaneous contractility in nonpregnant human myometrium. We also find that, while guanylyl cyclase serves as a target for CysNO in this tissue, increases in [cGMP]i are not required for CysNO-mediated relaxation; furthermore, application of cGMP analogs failed to alter myometrial contractility, evidence that PKG activation is not an important regulator of spontaneous contractility in nonpregnant human myometrium. A role for BK is suggested by our finding of a reversal of CysNO-mediated relaxations by pretreatment of tissues with charybdotoxin. Taken together, the results presented argue against a role for cGMP in the regulation of nonpregnant human uterine contractility. We have recently obtained preliminary evidence that argues against a role for cGMP in NO-induced relaxations in the term gestational (but not laboring) human myometrium; whether this is also the case in laboring tissue has not yet been determined.

    ACKNOWLEDGEMENTS

We thank Dr. David Harder for helpful conversations, Kirk Korver for developing the software used for integration, and the physicians and staff of the Reno Women's Center and Washoe County Medical Center for assistance in obtaining tissue samples.

    FOOTNOTES

This work was supported by National Institute of Child Health and Human Development Grants HD-33430 (M. E. Bradley) and HD-26227 (I. L. O. Buxton).

Current address of K. K. Bradley: Dept. of Pharmacology, Creighton University, 2500 California Plaza, Omaha, NE 68178.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. E. Bradley, Dept. of Pharmacology, Creighton University, 2500 California Plaza, Omaha, NE 68178.

Received 22 June 1998; accepted in final form 2 September 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

1.   Alonso-Galicia, M., H. A. Drummond, K. K. Reddy, J. R. Falck, and R. J. Roman. Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide. Hypertension 29: 320-325, 1997[Abstract/Free Full Text].

2.   Bolotina, V. M., S. Najibi, J. J. Palacino, P. J. Pagano, and R. A. Cohen. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850-853, 1994[Medline].

3.   Bradford, M. M. A rapid and sensitive assay for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

4.   Buhimschi, I., C. Yallampalli, Y. L. Dong, and R. E. Garfield. Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am. J. Obstet. Gynecol. 172: 1577-1584, 1995[Medline].

5.   Diamond, J. Lack of correlation between cyclic GMP elevation and relaxation of nonvascular smooth muscle by nitroglycerin, nitroprusside, hydroxylamine and sodium azide. J. Pharmacol. Exp. Ther. 225: 422-426, 1983[Abstract].

6.   Diamond, J. beta -Adrenoceptors, cyclic AMP, and cyclic GMP in control of uterine motility. In: Uterine Function, edited by M. E. Carsten, and J. D. Miller. New York: Plenum, 1989, p. 249-275.

7.   Diamond, J., and T. G. Holmes. Effects of potassium chloride and smooth muscle relaxants on tension and cyclic nucleotide levels in rat myometrium. Can. J. Physiol. Pharmacol. 53: 1099-1107, 1975[Medline].

8.   Figueroa, J. P., and G. A. Massmann. Estrogen increases nitric oxide synthase activity in the uterus of nonpregnant sheep. Am. J. Obstet. Gynecol. 173: 1539-1545, 1995[Medline].

9.   Franchi, A. M., M. Chaud, V. Rettori, A. Suburo, S. M. McCann, and M. Gimeno. Role of nitric oxide in eicosanoid synthesis and uterine motility in estrogen-treated rat uteri. Proc. Natl. Acad. Sci. USA 91: 539-543, 1994[Abstract].

10.   Garg, U. C., and A. Hassid. Nitric oxide decreases cytosolic free calcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism. J. Biol. Chem. 266: 9-12, 1991[Abstract/Free Full Text].

11.   Gibson, A., R. Babbedge, S. R. Brave, S. L. Hart, A. J. Hobbs, J. F. Tucker, P. Wallace, and P. K. Moore. An investigation of some S-nitrosothiols, and of hydroxy-arginine, on the mouse anococcygeus. Br. J. Pharmacol. 107: 715-721, 1992[Abstract].

12.   Gupta, S., C. McArthur, C. Grady, and N. B. Ruderman. Stimulation of vascular Na+-K+-ATPase activity by nitric oxide: a cGMP-independent effect. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2146-H2151, 1994[Abstract/Free Full Text].

13.   Harder, D. R., A. R. Lange, D. Gebremedhin, E. K. Birks, and R. J. Roman. Cytochrome P450 metabolites of arachidonic acid as intracellular signaling molecules in vascular tissue. J. Vasc. Res. 34: 237-243, 1997[Medline].

14.   Hennan, J. K., and J. Diamond. Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP. Br. J. Pharmacol. 123: 959-967, 1998[Abstract].

15.   Izumi, H., and R. E. Garfield. Relaxant effects of nitric oxide and cyclic GMP on pregnant rat uterine longitudinal smooth muscle. Eur. J. Obstet. Gynecol. Reprod. Biol. 60: 171-180, 1995[Medline].

16.   Izumi, H., C. Yallampalli, and R. E. Garfield. Gestational changes in L-arginine-induced relaxation of pregnant rat and human myometrial smooth muscle. Am. J. Obstet. Gynecol. 169: 1327-1337, 1993[Medline].

17.   Javid, P. J., S. W. Watts, and R. C. Webb. Inhibition of nitric oxide-induced vasodilation by gap junction inhibitors: a potential role for a cGMP-independent nitric oxide pathway. J. Vasc. Res. 33: 395-404, 1996[Medline].

18.   Jiang, H., J. L. Colbran, S. H. Francis, and J. D. Corbin. Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries. J. Biol. Chem. 267: 1015-1019, 1992[Abstract/Free Full Text].

19.   Kuenzli, K. A., M. E. Bradley, and I. L. O. Buxton. Cyclic GMP is not required for nitric oxide-induced relaxation in uterine smooth muscle. Br. J. Pharmacol. 119: 737-743, 1996[Abstract].

20.   Kuenzli, K. A., I. L. O. Buxton, and M. E. Bradley. Nitric oxide regulation of monkey myometrial contractility. Br. J. Pharmacol. 124: 63-68, 1998[Abstract].

21.   Levy, B., and B. E. Wilkenfeld. The potentiation of rat uterine inhibitory responses to noradrenaline by theophylline and nitroglycerin. Br. J. Pharmacol. 34: 604-612, 1968[Medline].

22.   Lloyd-Jones, D. M., and K. D. Bloch. The vascular biology of nitric oxide and its role in atherogenesis. Annu. Rev. Med. 47: 365-375, 1996[Medline].

23.   Maehara, T., A. Fujita, N. Suthamnatpong, T. Takeuchi, and F. Hata. Differences in relaxant effects of cyclic GMP on skinned muscle preparations from the proximal and distal colon of rats. Eur. J. Pharmacol. 261: 163-170, 1994[Medline].

24.   Muller, B., A. L. Kleschyov, S. Malblanc, and J. C. Stoclet. Nitric oxide-related cyclic GMP-independent relaxing effect of N-acetylcysteine in lipopolysaccharide-treated rat aorta. Br. J. Pharmacol. 123: 1221-1229, 1998[Abstract].

25.   Raber, G., S. Waldegger, T. Herzer, E. Gulbins, H. Murer, A. E. Busch, and F. Lang. The nitroso-donor S-nitroso-cysteine regulates IsK expressed in Xenopus oocytes via a cGMP independent mechanism. Biochem. Biophys. Res. Commun. 207: 195-201, 1995[Medline].

26.   Sandberg, M., V. Natarajan, I. Ronander, D. Kalderon, U. Walter, S. M. Lohmann, and T. Jahnsen. Molecular cloning and predicted full-length amino acid sequence of the type I isozyme of cGMP-dependent protein kinase from human placenta. FEBS Lett. 255: 321-329, 1989[Medline].

27.   Sciorati, C., G. Nistico, J. Meldolesi, and E. Clementi. Nitric oxide effects on cell growth: GMP-dependent stimulation of the AP-1 transcription complex and cyclic GMP-independent slowing of cell cycling. Br. J. Pharmacol. 122: 687-697, 1997[Abstract].

28.   Sekhar, K. R., R. J. Hatchett, J. B. Shabb, L. Wolfe, S. H. Francis, J. N. Wells, B. Jastorff, E. Butt, M. M. Chakinala, and J. D. Corbin. Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type Ialpha , compared with type I, cGMP-dependent protein kinase. Mol. Pharmacol. 42: 103-108, 1992[Abstract].

29.   Sladek, S. M., R. R. Magness, and K. P. Conrad. Nitric oxide and pregnancy. Am. J. Physiol. 272 (Regulatory Integrative Comp. Physiol. 41): R441-R463, 1997[Abstract/Free Full Text].

30.   Suthamnatpong, N., T. Maehara, A. Kanada, T. Takeuchi, and F. Hata. Dissociation of cyclic GMP level from relaxation of the distal, but not the proximal colon of rats. Jpn. J. Pharmacol. 62: 387-393, 1993[Medline].

31.   Suzuki, T., N. Nakajima, K. Fujimoto, T. Fujii, and K. Kawashima. Nitric oxide increases stimulation-evoked acetylcholine release from rat hippocampal slices by a cyclic GMP-independent mechanism. Brain Res. 760: 158-162, 1997[Medline].

32.   Takeuchi, T., M. Kishi, T. Ishii, H. Nishio, and F. Hata. Nitric oxide-mediated relaxation without concomitant changes in cyclic GMP content of rat proximal colon. Br. J. Pharmacol. 117: 1204-1208, 1996[Abstract].

33.   Word, R. A., M. L. Casey, K. E. Kamm, and J. T. Stull. Effects of cGMP on [Ca2+]i, myosin light chain phosphorylation, and contraction in human myometrium. Am. J. Physiol. 260 (Cell Physiol. 29): C861-C867, 1991[Abstract/Free Full Text].

34.   Yallampalli, C., R. E. Garfield, and M. Byam-Smith. Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinology 133: 1899-1902, 1993[Abstract].

35.   Yallampalli, C., H. Izumi, M. Byam-Smith, and R. E. Garfield. An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am. J. Obstet. Gynecol. 170: 175-185, 1994[Medline].

36.   Zou, A. P., J. T. Fleming, J. R. Falck, E. R. Jacobs, D. Gebremedhin, D. R. Harder, and R. J. Roman. 20-Hydroxyeicosatetraenoic acid is an endogenous inhibitor of the large conductance Ca2+-activated K+-channel in renal arterioles. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F228-F237, 1996.


Am J Physiol Cell Physiol 275(6):C1668-C1673
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