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Endocrinology Vol. 139, No. 5 2265-2271
Copyright © 1998 by The Endocrine Society

ARTICLES

Evidence for Inhibition by Protein Kinase A of Receptor/G{alpha}q/Phospholipase C (PLC) Coupling by a Mechanism Not Involving PLCß21

Kimberly L. Dodge and Barbara M. Sanborn

Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030

Address all correspondence and requests for reprints to: Barbara M. Sanborn, Ph.D., Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, P.O. Box 20708, Houston, Texas 77225. E-mail: bsanborn{at}utmmg.med.uth.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1–41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1–41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT-cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1–41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since Gß{gamma} activation of phospholipase Cß2 (PLCß2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCß2 via G{alpha}iGß{gamma} activation was explored. Western blot analysis of PHM1–41cells and COS-M6 cells detected PLCß1 and PLCß3, but not PLCß2. In PHM1–41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/G{alpha}q-coupled stimulation of PLCß1 or PLCß3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCß2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONTRACTION of smooth muscle is dependent on an increase in the concentration of intracellular free calcium. The uterine contractant oxytocin binds to the myometrial oxytocin receptor, resulting in the activation of a heterotrimeric G protein complex containing proteins of the G{alpha}q subfamily (1, 2) (subsequently designated as G{alpha}q). G{alpha}q, in turn, stimulates phospholipase C (PLC) activity of the PLCß subclass (3). PLC increases phosphatidylinositide turnover and the generation of inositol 1,3,4-trisphosphate (IP3), which releases calcium from internal stores (4).

Four types of PLCß (PLCß1–4) are stimulated through G protein activation. The G protein activated by a particular receptor dictates the PLCß subtype activated. PLCß1 is activated primarily by G{alpha}q subunits, while PLCß2 is stimulated predominately by Gß{gamma}-subunits (5, 6). Both G{alpha}q subunits and Gß{gamma} subunits have been demonstrated to stimulate PLCß3 (5). PLCß4 is located exclusively in the retina and probably does not stimulate phosphatidylinositide turnover in the uterus (7).

Sutherland and Rall (8) originally suggested that an elevation in cAMP correlated with the relaxation of smooth muscle. However, this relationship has since been challenged. Agonist-stimulated phosphatidylinositide turnover accompanies contraction, but reported effects of cAMP on phosphatidylinositide turnover range from stimulation to inhibition (9, 10, 11). We have previously found in the estrogen-primed rat myometrium that the uterine relaxants isoproterenol and relaxin, as well as 8-(-4 chlorophenylthio)(CPT)-cAMP, attenuated oxytocin-stimulated increases in both phosphatidylinositide turnover and intracellular free calcium concentrations (12, 13). The addition of H-8 at a concentration sufficient to inhibit cAMP-dependent protein kinase (PKA) blocked these effects (13). The data suggested that these relaxants, by increasing intracellular cAMP, inhibited the oxytocin-stimulated increase in phosphatidylinositide turnover via the action of PKA. In contrast to these findings, low concentrations of isoproterenol did not increase cAMP in the pregnant rat myometrium (14). However, isoproterenol still attenuated the increase in phosphatidylinositide turnover elicited by oxytocin, suggesting a possible cAMP-independent mechanism (14). Furthermore, forskolin did not attenuate the effect of oxytocin although it increased the concentration of cAMP in this tissue (14). Forskolin also did not inhibit the ability of oxytocin to stimulate phosphatidylinositide turnover in human uterine myocytes (15), suggesting that the cAMP-mediated inhibitory mechanism might not pertain in the human myometrium.

The reasons for these apparent discrepancies are not clear at present. They could reflect differences in experimental design or in species- or pregnancy-associated differences in the expression of regulatory or regulated proteins. In particular, a specific subtype of PLCß activated by receptor/G protein/PLC coupling (PLCß2) has recently been implicated in determining sensitivity to cAMP (16). However, several signaling pathways can result in PLC activation, and the pathways that are sensitive to cAMP in myometrium need to be defined.

The present study was designed to address a number of questions relating to inhibition by cAMP of the oxytocin-stimulated increase in phosphatidylinositide turnover in the uterus. We use an immortalized cell line derived from pregnant human myometrium (PHM1–41) (17) to address the presence of the cAMP-inhibitory mechanism in the human uterus and to explore the signaling pathway used by oxytocin. We also use COS-M6 cells transiently expressing G protein-coupled receptors to explore the generality of the inhibition by cAMP, hormones, and PKA itself in different cellular contexts. The nature of the G proteins activated by the oxytocin receptor and the PLCs stimulated are further delineated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[3H]myoinositol (22.3 Ci/mmol) was obtained from Dupont-New England Nuclear (Boston, MA). Pertussis toxin, CPT-cAMP, oxytocin, endothelin-1, norephinephrine, carbachol, forskolin, and isoproterenol were obtained from Sigma Chemical Co. (St. Louis, MO), and Rp-cAMPS was from Research Biochemicals International (Natick, MA). Antibodies against PLCß1, PLCß2, and PLCß3 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antirabbit IgG horseradish peroxidase conjugate was obtained from Bio-Rad (Hercules, CA). A vector expressing the human oxytocin receptor (pOTR) was provided by Dr. M. J. Brownstein (NIMH, Bethesda, MD). Vectors expressing G{alpha}q (pG{alpha}q) and the muscarinic M1 receptor (pM1R) were obtained from Dr. M. I. Simon (California Institute of Technology, Pasadena, CA), and a vector expressing the PKA catalytic subunit (PKA) was provided by Dr. G. S. McKnight (University of Washington, Seattle, WA). Cell culture reagents and lipofectamine were obtained from GIBCO BRL (Gaithersburg, MD). H-89 was obtained from Seikagaku America, Inc. (Rockville, MD). The DDT1 MF-2 cell line derived from hamster vas deferens was obtained from Dr. D. Lamb (Baylor College of Medicine, Houston, TX), and the HL-60 cell line used for the PLCß2 standard was obtained from Dr. M. C. Farach-Carson (University of Texas Houston Dental School, Houston, TX). Porcine relaxin was purified as described previously (18).

Cell culture
The immortalized pregnant human myometrial cell line PHM1–41 has been characterized elsewhere (17). Cells were cultured at 37 C and 5% CO2 in DMEM containing 4 g/liter glucose, 0.1 mg/ml Geneticin, 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin and was used at passages 16–20. DDT1 MF-2 and HL-60 cells were grown in the same medium in the absence of Geneticin. The medium for COS-M6 cells contained 8% FBS.

Transfection of COS-M6 cells
COS-M6 cells were plated in 35-mm plates in 1-ml aliquots at a density of 1.8 x 105 cells/ml. The next day they were incubated for 4 h with plasmids expressing the human oxytocin receptor (0.5 µg pOTR) or muscarinic M1 receptor (0.3 µg pM1R) and G{alpha}q (0.02 µg pG{alpha}q) in 400 µl DMEM with no FCS and 10 µl lipofectamine. After the incubation period, 500 µl of DMEM containing 20% FCS were added. Medium was changed 18 h later, and [3H]myoinositol was added 8 h later.

Phosphatidylinositide turnover
Cultured cells were incubated for 15–18 h in DMEM containing 0.4 µM [3H]myoinositol. Where indicated, the cells were then incubated in pertussis toxin (0.3 µg/ml) for 3 h. The cells were incubated with the indicated amounts of H-89 for 1 h or Rp-adenosine 3',5'-monophosphothioale triethylamine (Rp-cAMPS) for 20 min, followed by 10 mM LiCl for 10 min. Agents that elevate cAMP were added 15 (relaxin, forskolin, and isoproterenol) and 5 (CPT-cAMP) min before stimulation with agonist. At the times indicated, reactions were terminated by aspiration and the addition of 0.6 ml of cold 10% trichloroacetic acid. [3H]Inositol phosphates were isolated and counted essentially as described by Anwer et al. (12). Data represent mean ± SE of three determinations and were analyzed by one-way ANOVA and Duncan’s modified multiple range test.

Immunoblot analysis
Protein (30 µg) from whole cell lysates of PHM1–41, COS-M6, or HL-60 cells was subjected to SDS-PAGE in 5% gels and transferred to nitrocellulose membrane filters (Millipore Corp., Bedford, MA). Blots were probed with antibodies, and bands were visualized by enhanced chemiluminescence (DuPont-New England Nuclear, Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CPT-cAMP and uterine relaxants inhibit oxytocin-stimulated phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line
The effect of CPT-cAMP on oxytocin-stimulated phosphatidylinositide turnover was investigated in the immortalized pregnant human myometrial cell line PHM1–41. Stimulation with 100 nM oxytocin resulted in a 5.2-fold increase in [3H]IP3 (Fig. 1Go). Pretreatment of the cells with 0.9 mM CPT-cAMP had no effect on basal phosphatidylinositide turnover but inhibited the oxytocin-stimulated increase by 95%. The inhibition by CPT-cAMP was completely reversed by pretreatment for 1 h with 30 µM H-89, a protein kinase inhibitor with a high degree of specificity for PKA (19). H-89 alone had no significant effect on oxytocin-stimulated phosphatidylinositide turnover. The same general pattern was observed for total [3H]inositol phosphates, where oxytocin elicited a 4.0-fold increase, CPT-cAMP decreased it by 99%, and H-89 reversed the inhibition by 99% (data not shown). Additional experiments showed that 30 µM H-89 reversed the inhibition by 1.5, 1.2, and 0.9 mM CPT-cAMP by 79%, 88%, and 95%, respectively. Thus a relative balance between effects of PKA activation and inhibition was achieved over these concentration ranges.



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  		Figure 1. H-89 reversed the attenuation by CPT-cAMP of the oxytocin-induced increase in [3H]IP3 in PHM1–41 cells. Cells were incubated with 30 µM H-89 for 1 h, followed by CPT-cAMP (CPT) for 5 min, before being stimulated with 100 nM oxytocin (OT) for 10 min. Data are expressed as mean ± SE (n = 3) from a single experiment and are representative of three different experiments. Significant differences at P < 0.05 between groups are designated by different lowercase letters.

 
To substantiate the specificity of the kinase involved in the cAMP-inhibitory mechanism, the competitive cAMP antagonist Rp-cAMPS (20) was used to inhibit PKA activity. In an experiment similar to that in Fig. 1Go, the addition of 1.5 mM Rp-cAMPS to PHM1–41 cells reversed the inhibition by CPT-cAMP of oxytocin-stimulated phosphatidylinositide turnover by 88% (data not shown).

Relaxin is a uterine relaxant known to increase cAMP and to inhibit the oxytocin-induced increase in phosphatidylinositide turnover in the rat myometrium (12, 18). Forskolin is a direct activator of adenylyl cyclase activity (21). Figure 2Go shows that both relaxin and forskolin exerted inhibitory effects on phosphatidylinositide turnover in PHM1–41 cells, and that these effects were also attenuated by H-89. Forskolin (0.8 µM) inhibited the oxytocin-stimulated increase in phosphatidylinositide turnover by 94%, and this inhibition was reversed 82% by pretreatment with 30 µM H-89. Relaxin (1 µg/ml) also inhibited the oxytocin-stimulated increase by 92%, and the inhibition was reversed by 89% by H-89. Therefore, in this immortalized pregnant human myometrial cell line, CPT-cAMP, forskolin, and relaxin all inhibited phosphatidylinositide turnover, and the inhibition was reversed in each case by H-89.



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  		Figure 2. H-89 reversed the inhibition by forskolin and relaxin of the oxytocin-induced increase in [3H]IP3 in PHM1–41 cells. Cells were incubated with 30 µM H-89 for 1 h, followed by 0.8 µM forskolin (F) or 1 µg/ml relaxin (R) for 15 min, before being stimulated with 100 nM oxytocin (OT) for 10 min. Data are expressed as mean ± SE (n = 3) from a single experiment and are representative of two different experiments. Significant differences at P < 0.05 between groups are designated by different lowercase letters.

 
The inhibition of phosphatidylinositide turnover by cAMP is neither receptor nor cell specific
We have previously reported that PKA inhibited GTP{gamma}S-stimulated PLC activity in rat myometrial plasma membranes (22). This suggests that PKA phosphorylation of plasma membrane proteins may affect the coupling of G proteins to PLC independent of effects on the hormone receptor. To test whether the PKA pathway affected PLC stimulation by another hormone in PHM1–41 cells, the effect of CPT-cAMP on the endothelin-1-stimulated increase in phosphatidylinositide turnover was examined. Endothelin-1 is a uterine contractant that binds to the endothelin A receptor and stimulates G{alpha}q coupling to PLC (23). As shown in Fig. 3Go, treatment with 50 µM endothelin-1 for 10 min resulted in a 3.6-fold increase in [3H]IP3. Pretreatment with 1.5 mM CPT-cAMP inhibited the endothelin-1-stimulated increase by 99%, and H-89 attenuated the effect of CPT-cAMP by 74%. This pattern was also seen with total [3H]inositol phosphates. Endothelin-1 elicited a 2.9-fold increase, CPT-cAMP treatment inhibited the increase by 85%, and H-89 reversed the inhibition by 85%.



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  		Figure 3. Effect of CPT-cAMP on endothelin-1-stimulated increases in phosphatidylinositide turnover. PHM1–41 cells were incubated with 30 µM H-89, followed by 1.5 mM CPT-cAMP (CPT), before stimulation with 50 µM endothelin-1 (ET-1) for 10 min. Treatment with CPT-cAMP or H-89 alone resulted in no significant difference from the control. Data are expressed as mean ± SE (n = 3) from a single experiment and are representative of three different experiments. Significant differences at P < 0.05 between groups are designated by different lowercase letters.

 
To determine whether the oxytocin-stimulated increase in phosphatidylinositide turnover is sensitive to cAMP in cells not normally responding to oxytocin, COS-M6 cells were transiently transfected with plasmids expressing the oxytocin receptor and G{alpha}q, and the effect of CPT-cAMP on oxytocin-PLC coupling was determined. Figure 4Go shows that 100 nM oxytocin increased [3H]IP3 5.0-fold over basal activity in these transfected cells, and preincubation with 1.2 mM CPT-cAMP inhibited this increase by 99%. The addition of 10 µM H-89 reversed the inhibition caused by CPT-cAMP by 97%. In other experiments, treatment with CPT-cAMP or 10 µM H-89 alone resulted in no significant difference from the control, whereas 30 µM H-89 increased basal phosphatidylinositide turnover. Inhibition by 1.5 mM CPT-cAMP was reversed 80% by 10 µM H-89.



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  		Figure 4. H-89 reversed the attenuation by CPT-cAMP of the oxytocin-induced increase in [3H]IP3 in COS-M6 cells. Cells were transiently transfected with plasmids expressing pOTR and pG{alpha}q. Cells were incubated with 10 µM H-89 for 1 h, followed by 1.2 mM CPT-cAMP (CPT) for 5 min, before stimulation with 100 nM oxytocin (OT) for 30 min. Data are expressed as mean ± SE (n = 3) from a single experiment and are representative of three different experiments. Significant differences at P < 0.05 between groups are designated by different lowercase letters.

 
Endogenously generated increases in cAMP also inhibited oxytocin-dependent phosphatidylinositide turnover in COS-M6 cells transiently expressing the oxytocin receptor. Isoproterenol activates adenylate cyclase, resulting in an increase in cAMP concentration in COS cells (24). Activation of ß-adrenergic receptors with 20 nM isoproterenol inhibited the oxytocin-stimulated increase in phosphatidylinositide turnover by 100%, and the inhibition was reversed 100% by pretreatment with 10 µM H-89 (data not shown). CPT-cAMP also inhibited phosphatidylinositide turnover stimulated by norephinephrine (a G{alpha}q-coupled receptor) (25) by 99% in DDT1 MF-2 smooth muscle cells (data not shown).

Evidence against oxytocin receptor-stimulated activation of PLCß2 in PHM1-41 and COS-M6 cells
Liu and Simon (16) have reported that Gß{gamma} subunit activation of overexpressed PLCß2 is inhibited by PKA in COS-M6 cells. However, in these cells, endogenous PLCß1 and PLCß3 activities were apparently not affected by PKA. If this finding is a general one, it predicts that only receptors that stimulate primarily PLCß2 would exhibit a decrease in phosphatidylinositide turnover in the presence of cAMP. While Western blot analysis performed on whole cell lysates of PHM1–41 cells and COS-M6 cells revealed PLCß1 and PLCß3 in both cell types, no significant amount of PLCß2 was observed in either cell type (Fig. 5Go). PLCß2 was present in HL-60 cells, however. Thus, PKA inhibition that targets primarily PLCß2 seems unlikely in either PHM1–41 or COS-M6 cells.



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  		Figure 5. Detection of PLCß1 and PLCß3, but little PLCß2, in whole cell lysates of PHM1–41 cells and COS-M6 cells. Immunoblots were probed with antibodies specifically directed against PLCß1, PLCß2, and PLCß3 (1:400 each). HL-60 cells were used as a positive control for PLCß2.

 
Inhibition by cAMP of oxytocin receptor/G{alpha}q/PLCß1 or PLCß3 coupling contradicts the finding of Liu and Simon that only PLCß2 activity was inhibited by PKA (16). These authors observed no effect of PKA catalytic subunit on stimulation by carbachol of endogenous PLCß1 and PLCß3 activity in COS-7 cells overexpressing the muscarinic M1 receptor and the G{alpha}q isoforms G{alpha}15 and G{alpha}16. To explore the source of this discrepancy, COS-M6 cells were transfected with plasmids expressing the muscarinic M1 receptor and G{alpha}q. Figure 6Go shows that treatment with 15 µM carbachol resulted in a 3.4-fold increase in [3H]IP3. Treatment with 1.2 mM CPT-cAMP inhibited the effect of carbachol by 97%, and this inhibition was completely reversed by pretreatment with H-89. Importantly, cotransfection of a plasmid expressing the PKA catalytic subunit also inhibited the carbachol-stimulated increase in phosphatidylinositide turnover by 96%. Similarly, the PKA catalytic subunit inhibited the oxytocin-induced increase in phosphatidylinositide turnover by 89% in COS-M6 cells expressing the oxytocin receptor. These data are consistent with an inhibition of phosphatidylinositide turnover via the action of PKA. They demonstrate that the cAMP-inhibitory mechanism affects phosphatidylinositide turnover stimulated by both the oxytocin and muscarinic M1 receptors in COS-M6 cells, independent of effects on PLCß2.



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  		Figure 6. PKA inhibits the ability of carbachol to increase [3H]IP3 in COS-M6 cells. Cells were transiently transfected with plasmids expressing pM1R and pG{alpha}q. Cells were incubated with 10 µM H-89 for 1 h, followed by 1.2 mM CPT-cAMP (CPT) for 5 min, before stimulation with 15 µM carbachol (M1R) for 30 min. Cells transfected with plasmids expressing PKA catalytic subunit were exposed to 60 µM ZnSO4 overnight to stimulate expression. Data are expressed as mean ± SE (n = 3) from a single experiment and are representative of three different experiments. Significant differences at P < 0.05 between groups are designated by different lowercase letters.

 
The action of pertussis toxin is consistent with a PKA-mediated inhibition of phosphatidylinositide turnover
Gß{gamma} subunits have been demonstrated to stimulate PLCß3 activity as well as PLCß2 (5). Gß{gamma} subunits could be released from either receptor-activated pertussis toxin-sensitive or -insensitive G{alpha} subunits, namely G{alpha}i/G{alpha}o and G{alpha}q, respectively. Although the stimulation of phosphatidylinositide turnover by oxytocin has been demonstrated to occur through oxytocin receptor/G{alpha}q coupling (1, 2), Strakova and Soloff (26) have recently reported that G{alpha}i subunits also associate with the oxytocin receptor in pregnant rat myometrial plasma membrane extracts. Pertussis toxin ADP-ribosylates G{alpha}i subunits, rendering them inactive and unable to dissociate from Gß{gamma} (27). Pertussis toxin treatment can partially or totally inhibit the oxytocin-stimulated increase in phosphatidylinositide turnover in myometrium from several species (12, 15, 28, 29). Preincubation with 0.3 µg/ml pertussis toxin decreased the increase in [3H]IP3 elicited by oxytocin by 53% (Fig. 7Go). Pertussis toxin alone had no effect on basal [3H]IP3.



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  		Figure 7. Effect of pertussis toxin treatment on phosphatidylinositde turnover in PHM1–41 cells. PHM1–41 cells were incubated with 0.3 µg/ml pertussis toxin (PTX) for 3 h. H-89 (30 µM) was added in the last hour. Cells were then stimulated with 100 nM oxytocin (OT) for 10 min. H-89 alone did not increase [3H]IP3. Data are expressed as mean ± SE (n = 3) from a single experiment and are representative of two different experiments. Significant differences at P < 0.05 between groups are designated by different lowercase letters.

 
One interpretation of these data could be that a portion of the oxytocin-stimulated increase involves the activation of G{alpha}i and stimulation of PLCß3 by the released Gß{gamma}. However, we have found evidence for an alternative mechanism. In the rat myometrium, the inhibitory effect of pertussis toxin was reversed by the protein kinase inhibitor H-8 (28). These data suggested that inhibition of G{alpha}i by pertussis toxin released the inhibitory influence of G{alpha}i on adenylyl cyclase, resulting in an increase in cAMP and activation of PKA. As shown above, PKA can have an inhibitory effect on the coupling of G{alpha}q to PLC. To determine whether such a mechanism pertains in PHM1–41 cells, they were treated with pertussis toxin in the absence and presence of H-89 and then stimulated by oxytocin. Figure 7Go shows that 30 µM H-89 completely reversed the inhibitory effect of pertussis toxin. H-89 had no effect on the ability of oxytocin to stimulate [3H]IP3. These data are consistent with an inhibitory mechanism on PLC activation involving an increase in cAMP and activation of PKA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, CPT-cAMP, relaxin, and forskolin all inhibited the oxytocin-stimulated increase in phosphatidylinositide turnover in the immortalized PHM1–41 cell line derived from pregnant human myometrium, and the protein kinase inhibitor H-89 reversed the inhibition. CPT-cAMP also inhibited phosphatidylinositide turnover stimulated by endothelin-1 in the PHM1–41 cells. These data suggest that cAMP-mediated inhibition of phosphatidylinositide turnover through the action of PKA has potential importance in human myometrium.

Although we have demonstrated that the cAMP-inhibitory effect on phosphatidylinositide turnover can be important in the myometrium, it is not universally observed. Khac et al. (14) found that forskolin did not inhibit the oxytocin-stimulated increase in the pregnant rat uterus, despite an elevation in cAMP levels. Phaneuf et al. (15) reported the insensitivity of the oxytocin-stimulated increase in phosphatidylinositide turnover to forskolin in human uterine myocytes and suggested the cAMP-inhibitory mechanism did not pertain to the human myometrium. In contrast, we observed effects of CPT-cAMP and forskolin in both the human myometrial cell line and in the unrelated COS-M6 cells, suggesting that a factor peculiar to the myometrial phenotype is probably not responsible for the effect. The basis for the disparate effects of forskolin are not clear, since similar concentrations of forskolin were used in these studies, and no major differences in methodology were obvious. While it is possible that immortalization has changed the phenotype of the PHM1–41 cell line, these cells retain several phenotypic characteristics of the myometrium, including oxytocin receptors, and respond to oxytocin in a similar fashion as uterine tissue and cells with regard to increases in phosphatidylinositide turnover and intracellular calcium (17). The PHM1–41 cell line was derived from a single patient, and variation between responses in cells from different patients could possibly explain the differences observed.

Support for the generality of the cAMP-inhibitory mechanism derives from the observation that CPT-cAMP inhibited phosphatidylinositide turnover in COS-M6 cells and DDT1 MF-2 cells as well as PHM1–41 cells. Furthermore, CPT-cAMP inhibited the increase in phosphatidylinositide turnover stimulated by oxytocin and carbachol in COS-M6 cells transfected with plasmids expressing the respective receptors and the norepinephrine-stimulated increase in DDT1 MF-2 cells. In COS-M6 cells overexpressing either the oxytocin receptor or muscarinic M1 receptor and G{alpha}q, PKA catalytic subunit also inhibited the ability of both agonists to stimulate phosphatidylinositide turnover. This evidence supports the conclusion that the PKA-mediated inhibition of agonist-stimulated phosphatidylinositide turnover could pertain to different receptors and tissue types.

Liu and Simon (16) reported that PKA inhibited PLCß2 activation by Gß{gamma}, but not ligand-dependent activation of endogenous PLC activity attributable to PLCß1 and PLCß3 in COS-7 cells. However, the data presented here indicate that the cAMP-inhibitory mechanism can target proteins other than PLCß2. Western blot analysis showed no significant amount of PLCß2 in either the PHM1–41 or the COS-M6 cells. However, we clearly demonstrate the inhibition of agonist-stimulated increases in phosphatidylinositide turnover by cAMP in both of these cell lines although no PLCß2 is present. Moreover, in contrast to the results of Liu and Simon in COS-7 cells (16), we found that CPT-cAMP and the catalytic subunit of PKA inhibited muscarinic M1 receptor stimulation of phosphatidylinositide turnover in COS-M6 cells in the absence of PLCß2. The studies performed by Liu and Simon on the muscarinic M1 receptor used the G{alpha}q isoforms G{alpha}15 and G{alpha}16 while the studies described here examined coupling to G{alpha}q. It is possible that PKA may not inhibit receptor-stimulated coupling of G{alpha}15 and G{alpha}16 isoforms to PLCs. Although COS-M6 cells are a subtype of COS cells, the differences in response could also be due to some other alteration between these cells.

The finding that the oxytocin-stimulated increase in phosphatidylinositide turnover is only partially sensitive to pertussis toxin in the PHM1–41 cells and in myometrial cells (12, 15, 28, 29) indicates that coupling of the oxytocin receptor to endogenous G{alpha}i in the uterus is not obligatory for this action. In support of this finding, we have been able to completely inhibit the oxytocin-stimulated increase in phosphatidylinositide turnover and oxytocin-stimulated GTPase activity in myometrial membranes with anti-G{alpha}q/11 IgG (1). Arnaudeau et al. (2) also reported that an anti-G{alpha}q/11 antibody, delivered via pipette to a rat myometrial cell in the whole cell patch mode, inhibited the oxytocin-stimulated increase in intracellular calcium (2).

Although pertussis toxin sensitivity has been interpreted to indicate the involvement of G{alpha}i or G{alpha}o in receptor-effector coupling, we found in the rat myometrium that pertussis toxin can attenuate oxytocin-stimulated phosphatidylinositide turnover indirectly via activation of PKA (28). This is apparently the case in PHM1–41 cells as well, since the addition of the protein kinase inhibitor H-89 reversed the inhibitory effect of pertussis toxin on phosphatidylinositide turnover by 95%. These data provide additional support for the contention that in these cells, the oxytocin receptor coupling to PLC via a pertussis toxin-insensitive G protein of the G{alpha}q family is attenuated by the action of PKA.

The proteins phosphorylated by PKA that mediate its inhibitory effect on phosphatidylinositide turnover are not defined at present. Since GTP{gamma}S-stimulated PLC activity was inhibited by PKA in rat myometrial plasma membranes, Wen et al. (22) postulated that the inhibitory effect influenced G-protein/PLC coupling. We have demonstrated here the generality of cAMP inhibition of receptor/G{alpha}q/PLCß1 or PLCß3 coupling. However, we and others have not found phosphorylation of either G{alpha}q or PLCß1 (Ref. 30, and C. Yue and B. Sanborn, unpublished observations). There is increasing evidence for the influence of other proteins on G protein/PLC coupling (31, 32, 33), and one of these proteins may be a target of PKA phosphorylation. It seems plausible that the concentrations of these regulated proteins might be altered in different cell types, hormonal states, or species (34, 35, 36). This may, in part, explain the lack of correlation between an elevation in cAMP and inhibition of oxytocin-stimulated phosphatidylinositide turnover in the late pregnant rat myometrium (14).

In summary, we present data supporting a role for cAMP-stimulated PKA activation in the inhibition of receptor/G{alpha}q/PLC coupling that does not involve the phosphorylation of PLCß2. This mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation. In the pregnant human myometrial cell line PHM1–41, this inhibitory action probably influences oxytocin receptor/G{alpha}q/PLCß1 or PLCß3 coupling. This mechanism may contribute to the actions of hormones and tocolytics that attenuate uterine contractions.


   Acknowledgments
 
The authors wish to thank Dr. M. J. Brownstein for the expression vector expressing the human oxytocin receptor, Dr. M. I. Simon for the G{alpha}q and muscarinic M1 receptor vectors, Dr. G. S. McKnight for the vector expressing the PKA catalytic subunit, and Drs. D. Lamb and M. C. Farach-Carson for the DDT1 MF-2 and HL-60 cells, respectively.


   Footnotes
 
1 This work was supported in part by NIH Grants HD-09618 and T32-HD-07325. This work partially fulfills the requirements of a Ph.D. degree (for K.L.D.) by the Graduate School of Biomedical Sciences of the University of Texas Health Science Center-Houston. Back

Received August 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ku CY, Qian A, Wen Y, Anwer K, Sanborn BM 1995 Oxytocin stimulates myometrial guanosine triphosphatase and phospholipase-C activities via coupling to G{alpha}q/11. Endocrinology 136:1509–1515[Abstract]
  2. Arnaudeau S, Lepretre N, Mironneau J 1994 Oxytocin mobilizes calcium from a unique heparin-sensitive and thapsigargin-sensitive store in single myometrial cells from pregnant rats. Pflugers Arch 428:51–59[CrossRef][Medline]
  3. Lee CH, Park D, Wu D, Rhee SG, Simon MI 1992 Members of the Gq {alpha} subunit gene family activate phosphoipase C ß isozymes. J Biol Chem 267:16044–16047[Abstract/Free Full Text]
  4. Berridge MJ 1995 Inositol trisphosphate and calcium signaling. Ann NY Acad Sci 766:31–43[Medline]
  5. Smrcka AV, Sternweis PC 1993 Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C ß by G-protein {alpha} and ß{gamma} subunits. J Biol Chem 268:9667–9674[Abstract/Free Full Text]
  6. Katz A, Wu D, Simon MI 1992 Subunits beta gamma of heterotrimeric G protein activate beta 2 isoforms of phospholipase C. Nature 360:686–689[CrossRef][Medline]
  7. Lee CW, Park DJ, Lee KH, Kim CG, Rhee SG 1993 Purification, molecular cloning and sequencing of phospholipase-C beta 4. J Biol Chem 21:21318–27
  8. Sutherland EW, Rall TW 1960 The relation of adenosine-3',5'-phosphates and phosphorylase to the actions of catecholamines and other hormones. Pharmacol Rev 12:265–299[Free Full Text]
  9. Pittner RA, Fain JN 1989 Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production. Endocrinology 257:455–460
  10. Taguchi M, Field JB 1988 Effects of thyroid-stimulating hormone, carbachol, norepinephrine, and adenosine 3',5'-monophosphate on polyphosphatidylinositol phosphate hydrolysis in dog thyroid slices. Endocrinology 123:2019–26[Abstract/Free Full Text]
  11. Kato H, Ishitoya J, Takenawa T 1986 Inhibition of inositol phospholipids metabolism and calcium mobilization by cyclic AMP-increasing agents and phorbol ester in neutrophils. Biochem Biophys Res Commun 123:1272–78
  12. Anwer K, Hovington JA, Sanborn BM 1989 Antagonism of contractants and relaxants at the level of intracellular calcium and phosphoinositide turnover in the rat uterus. Endocrinology 124:2995–3002[Abstract/Free Full Text]
  13. Anwer K, Hovington JA, Sanborn BM 1990 Involvement of protein kinase A in the regulation of intracellular free calcium and phosphoinositide turnover in the rat myometrium. Biol Reprod 43:851–859[Abstract]
  14. Khac LD, Arnaudeau S, Lepretre N, Mironneau J, Harbon S 1996 Beta adrenergic receptor activation attenuates the generation of inositol phosphates in the pregnant rat myometrium. Correlation with inhibition of Ca++ influx, a cAMP-independent mechanism. J Pharmacol Exp Ther 276:130–136[Abstract/Free Full Text]
  15. Phaneuf S, Europe-Finner GN, Varney M, MacKenzie IZ, Watson SP, Lopez-Bernal A 1993 Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins. J Endocrinol 136:497–509[Abstract/Free Full Text]
  16. Liu M, Simon MI 1996 Regulation by cAMP-dependent protein kinase of a G-protein mediated phospholipase C. Nature 382:83–87[CrossRef][Medline]
  17. Monga M, Ku CY, Dodge K, Sanborn BM 1996 Oxytocin-stimulated responses in a pregnant human immortalized myometrial cell line. Biol Reprod 55:427–432[Abstract]
  18. Hsu CJ, McCormack SM, Sanborn BM 1985 The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in rat myometrial cells in culture. Endocrinology 116:2029–2035[Abstract/Free Full Text]
  19. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayash, K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272[Abstract/Free Full Text]
  20. Van Hasstert PJ, Van Driel R, Jastorff B, Baraniak J, Stec WJ, De Witt RJ 1984 Competitive cAMP antagonist for cAMP-receptor proteins. J Biol Chem 259:10020[Abstract/Free Full Text]
  21. Seamon KB, Daly JW 1986 Forskolin: its biological and chemical properties. Adv Cyclic Nucleotides Protein Phosphorylation Res 20:1–150[Medline]
  22. Wen Y, Anwer K, Singh SP, Sanborn BM 1992 Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes. Endocrinology 131:1377–1382[Abstract/Free Full Text]
  23. Khac LD, Naze S, Harbon S 1994 Endothelin receptor type A signals both the accumulation of inositol phosphates and the inhibition of cyclic AMP generation in rat myometrium: stimulation and desensitization. Mol Pharmacol 46:485–494[Abstract]
  24. Cotecchia S, Ostrowski J, Kjelsberg MA, Caron MG, Lefkowitz RJ 1991 Discrete amino acid sequences of the {alpha}1-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis. J Biol Chem 267:1633–1639[Abstract/Free Full Text]
  25. Schachter JB, Ivins JK, Pittiman RN, Wolfe BB 1992 Competitive regulation of phospholipase C responses by cAMP and calcium. Mol Pharmacol 41:577–586[Abstract]
  26. Strakova Z, Soloff MS 1997 Coupling of oxytocin receptors to G-proteins in rat myometrium during labor:Gi-receptor interactions. Am J Physiol 272:E870–E878
  27. De Vivo M, Gershengorn MC 1990 ADP-Ribosylating Toxins and G-Proteins. American Society of Microbiology, Washington DC, pp 267–293
  28. Singh SP, Anwer K, Wen Y, Sanborn BM 1992 Inhibition of oxytocin-stimulated phosphoinositide turnover in rat myometrium by pertussis and cholera toxins may involve protein kinase A activation. Cell Signal 4:619–625[CrossRef][Medline]
  29. Molnar M, Hertelendy F 1990 Regulation of intracellular free calcium in human myometrial cells by prostaglandin F2 alpha: comparison with oxytocin. J Clin Endocrinol Metab 71:1243–1250[Abstract/Free Full Text]
  30. Kim UH, Kim JW, Rhee SG 1989 Phosphorylation of phospholipase C-gamma by cAMP-dependent protein kinase. J Biol Chem 264:20167–20170[Abstract/Free Full Text]
  31. Sato M, Ribas C, Hildebrant JD, Lanier SM 1996 Characterization of a G-protein activator in the neuroblastoma-glioma cell hybrid NG108–15. J Biol Chem 271:30052–30060[Abstract/Free Full Text]
  32. Berman DM, Kozasa T, Gilman AG 1996 The GTPase-activation protein RGS4 stabilized the transition state for nucleotide hydrolysis. J Biol Chem 271:27209–27212[Abstract/Free Full Text]
  33. Noaoff C, Mitterauer T, Roka F, A membrane protein which impedes receptor-G protein signaling. Program of the 9th International Conference of Second Messengers and Phosphoproteins, 1997, Nashville, TN (Abstract 513)
  34. Europe-Finner AN, Phaneuf S, Watson SP, Lopez-Bernal A 1993 Identification and expression of G-proteins in human myometrium: up-regulation of G{alpha}s in pregnancy. Endocrinology 132:2484–2490[Abstract/Free Full Text]
  35. Cohen-Tannoudji J, Mhaouty S, Elwardy-Merezak J, Lecrivain JL, Robin MT, Legrand C, Maltier JP 1995 Regulation of myometrial Gi2, Gi3, and Gq expression during pregnancy. Effects of progesterone and estradiol. Biol Reprod 53:55–64[Abstract]
  36. Phillippe M, Harrison HH 1992 Gestational modulation of myometrial proteins in the timed-pregnant Sprague-Dawley rat. Life Sci 50:1189–1200[CrossRef][Medline]



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