Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells

Eun Sung Park1, Clement O. Echetebu1, Solweig Soloff1, and Melvyn S. Soloff1,2

1 Department of Obstetrics and Gynecology, 2 Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1062


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Regulators of G protein signaling (RGS proteins) interact with Galpha q and Galpha i and accelerate GTPase activity. These proteins have been characterized only within the past few years, so our understanding of their importance is still preliminary. We examined the effect of oxytocin on RGS2 mRNA expression to help determine the role of RGS proteins in oxytocin signaling in human myometrial cells in primary culture. Oxytocin increased RGS2 mRNA concentration maximally by 1 or 2 h in a dose-dependent and agonist-specific manner. RGS2 mRNA levels were also elevated by treatment with Ca2+ ionophore, phorbol ester, or forskolin. Oxytocin's effects were completely inhibited by an intracellular Ca2+ chelator and partially blocked by a protein kinase C inhibitor, indicating that intracellular Ca2+ concentration is the primary signal for oxytocin elevation of RGS2 mRNA levels. Use of pharmacological inhibitors indicated that part of oxytocin-stimulated RGS2 mRNA expression is mediated by Gi/tyrosine kinase activities. Although oxytocin does not stimulate increases in intracellular cAMP concentration, agents that elevate intracellular cAMP concentrations and cause myometrial relaxation may possibly cause heterologous desensitization to oxytocin via RGS2 expression. These results suggest that RGS2 may be important in regulating the myometrial response to oxytocin.

intracellular calcium; protein kinase C; G proteins; forskolin; adenosine 3',5'-cyclic monophosphate; regulator of G protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROTEINS TERMED REGULATORS of G protein signaling (RGS proteins) are a diverse multiprotein family that interacts with activated Galpha subunits to block signaling by Gi and/or Gq classes of G proteins (12). The RGS protein binding results in accelerated hydrolysis of Galpha -bound GTP. As part of the general class of GTPase-activating proteins, RGS proteins may be important for turning off many G protein-mediated physiological responses, accounting for attenuated responses or, in the extreme, what appears to be uncoupling of G protein activity from liganded receptors. GTPase-activating proteins have been characterized only within the past few years, so our understanding of their importance is still rudimentary. Several RGS mRNAs are constitutively expressed at high levels (8, 22), suggesting that the corresponding proteins might be readily available for the acute desensitization of signaling. In contrast, RGS2 mRNA levels are typically low in resting cells but are upregulated for several hours after stimulation by various agents in different cell types (11, 13, 14, 19, 22, 26). RGS2 mRNA is presumably synthesized in response to an initial stimulus and then blocks subsequent hormone-signaling events.

Oxytocin (OT) is a G protein-coupled receptor agonist that stimulates uterine smooth muscle contraction. Human myometrial cells in culture express OT receptors, and, although these cells are not in an appropriate environment to contract, they possess signal pathways involved in the stimulation of contraction. Thus myosin light chain kinase activity can be activated in myometrial smooth muscle cells by elevation of intracellular Ca2+ concentrations (2). OT also stimulates an increase in prostacyclin synthesis by a G protein-sensitive (pertussis toxin-inhibitable) pathway in cultured human myometrial cells (20). RGS2 presumably is expressed in all cell types, but nothing is known of its function in uterine smooth muscle cells. We have shown in the present studies that OT stimulates increased expression of RGS2 mRNA in human myometrial cells in primary culture and elucidated the major signal pathways involved.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Reagents were obtained from the following sources: OT and OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin from Peninsula Laboratories (Belmont, CA); pertussis toxin, GF-109203X, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), genestein, wortmannin, and phorbol 12-myristate 13-acetate (PMA) from Biomol Research Laboratories (Plymouth Meeting, PA); FBS from Atlanta Biological (Atlanta, GA); MEM and cell culture reagents from GIBCO-BRL (Grand Island, NY). All other chemicals were obtained from Sigma (St. Louis, MO).

Myometrial cell culture. The University of Texas Medical Branch Committee on Research Involving Human Subjects approved the use of human tissue. Myometrial samples were taken from women by caesarean section near the end of gestation, and cells were prepared as described previously (5). The cells were maintained in MEM containing 10% (vol/vol) FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin G, 100 µg/ml streptomycin sulfate, and 15 µg/ml amphotericin B at 37°C (95% humidity) in the presence of 5% CO2. The cells, which were used at confluence between passages 3 and 10, were serum starved overnight (about 16 h) before treatment with OT or other agents.

Northern blot analysis. Total RNA was isolated from cultured human myometrium cells using TRIzol reagent (GIBCO-BRL Life Technologies, Rockville, MD). Samples of 20 µg of RNA were subjected to electrophoresis in 1% agarose-formaldehyde gels and transferred to nylon membranes for Northern blotting. The RGS2 probe was obtained by RT-PCR and cloned into pGEM-T Easy (Promega, Madison, WI). We identified selected clones by restriction enzyme digestion and DNA sequencing. Hybridizations were performed using random primed cDNA fragments of the entire coding regions of RGS2 gene labeled with [alpha -32P]dCTP (3,000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) in ExpressHyb hybrization solution (Clontech Laboratories, Palo Alto, CA) overnight at 68°C. The blots were rinsed for 10 min at room temperature with 1× saline-sodium citrate (SSC) and 0.1% SDS solution and then 45 min at 65°C with 0.2× SSC and 0.1% SDS solution. The blots were exposed to a Cyclone phosphor screen (Packard Instrument, Meriden, CT) for image analysis and to X-ray film at -80°C for 1-3 days. After the films were developed, the nylon membranes were stripped and reprobed with the human alpha -tubulin cDNA, which was synthesized by RT-PCR, cloned, and labeled by random priming as described for the RGS2 probe.

Analysis of data. Experiments were repeated on myometrial cells derived from three separate women. The results of the Northern blots were analyzed densitometrically and quantified using ImageQuant software (Packard Instrument). In cases where a percent change is reported, the values were obtained by expressing the RGS2 mRNA concentration relative to that of alpha -tubulin mRNA. Because of variability in the results between cells in primary culture from different patients, the data are presented as a representative Northern blot from a triplicate set. The changes in RGS2 expression within each treatment group were comparable in all cases.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OT specifically increases RGS2 mRNA expression. Treatment of human myometrial cells with 100 nM OT resulted in an increase in the amount of RGS2 mRNA by 30 min (Fig. 1). Maximal stimulation was apparent by 1-2 h (Fig. 1). An effect of OT at 2 h was seen with as little as 0.1 nM OT, and 1 nM OT gave a near-maximal response (Fig. 2A). The effects of OT were blocked by the OT antagonist (Fig. 2B).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Northern blot analysis of the effects of oxytocin (100 nM) on regulator of G protein (RGS2) mRNA levels in cultured human myometrial cells. After analysis, the blots were reprobed to determine alpha -tubulin mRNA concentration, which was used to normalize RGS2 mRNA levels. alpha -TUB, alpha -tubulin.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 2.   Specificity of the RGS2 mRNA response to oxytocin (OT). A: dose-response relationship between OT concentration and RGS2 mRNA levels. Cells were treated with OT for 2 h. B: inhibition of the OT-stimulated RGS2 mRNA response by OT antagonist (OTA). Cells were treated either with 10 nM OT, 100 nM OT antagonist, or both for 2 h.

Mediators in OT-stimulated RGS2 mRNA expression. OT signaling in the myometrium has been shown to occur via Gq/phospholipase C (PLC) and activation of protein kinase C (PKC; see Refs. 9, 21, 24). Treatment of myometrial cells with the Ca2+ ionophore A-23187 (50 µM) to elevate intracellular Ca2+ concentrations resulted in an increase in RGS2 mRNA levels, with a maximal increase at ~2 h (Fig. 3A). A-23187 treatment, however, caused the degradation of mRNA at later time points, as was apparent by alpha -tubulin analysis (Fig. 3A). Increases in intracellular Ca2+ concentrations generated by OT treatment appear to be responsible for OT-stimulated expression of RGS2 mRNA, as pretreatment of cells with the Ca2+ chelator BAPTA-AM (10 and 30 µM) blocked the effects of OT (Fig. 3B). The effects of A-23187 treatment were also blocked by the two concentrations of BAPTA-AM (Fig. 3B).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 3.   Involvement of intracellular Ca2+ concentration on OT-stimulated RGS2 mRNA expression. A: effect of Ca2+ ionophore A-23187 after 2 h of treatment. B: effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) pretreatment on A-23187- and OT-stimulated RGS2 mRNA expression.

OT signaling through stimulation of PLC activity results in activation of PKC. Activation of PKC by PMA also stimulated an increase in RGS2 mRNA 2 h after treatment (Fig. 4). This effect was blocked by pretreatment with the PKC inhibitor GF-109203X, at 1 and 10 µM. The effects of OT on RGS2 mRNA levels, however, were only partially blocked by the same doses of GF-109203X.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA; 100 nM) and inhibition of PKC activity by GF-109203X (1 and 10 µM) on RGS2 mRNA expression. Cells were pretreated with inhibitor for 30 min, followed by treatment with either PMA or OT (100 nM) for 2 h.

Increases in intracellular cAMP have been shown to induce increases in RGS2 mRNA levels in certain cell types but not others. Elevation of intracellular cAMP concentration by forskolin treatment caused a marked increase in RGS2 mRNA levels (Fig. 5). Thus human myometrial cells are in the category of cell type that responds to cAMP with an increase in RGS2 mRNA expression.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of forskolin (FSK; 25 µM) on RGS2 mRNA expression in human myometrial cells.

Synergy between signal pathway activators on RGS2 expression. The effects of A-23187 and PMA were potentiated by cotreatment with forskolin (Fig. 6). Likewise, A-23187 synergized with PMA to enhance RGS2 mRNA expression. The effects of PMA in conjunction with either A-23187 or forskolin were notable in that PMA alone was not stimulatory at the time point taken. The findings indicate that RGS2 mRNA levels are regulated along separate and complementary pathways with respect to increased intracellular Ca2+ concentrations, activation of PKC, and elevation of intracellular cAMP levels.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   Synergistic effects of A-23187 (50 µM), forskolin (25 µM), and PMA (100 nM) on RGS2 mRNA expression. Cells were treated for 2 h.

Other potential pathways mediating OT stimulation of RGS2 mRNA expression. The effects of OT on stimulation of inositol trisphosphate and increased intracellular Ca2+ concentrations are mediated by both pertussis toxin-sensitive and -insensitive pathways (25). Pretreatment of human myometrial cells with pertussis toxin (either 200 or 400 ng/ml) for 18 h blunted the increase in RGS2 expression stimulated by 2 h of treatment with OT (Fig. 7). There was a 32-51% reduction in OT-stimulated RGS2 expression with pertussis toxin treatment. These findings indicate that a portion of the effects of OT on RGS2 mRNA are mediated by Gi/o. Agonists for receptors coupled to Gi/o have been shown to cross-talk through Gbeta gamma to nonreceptor and receptor tyrosine kinase pathways (17). In keeping with the pertussis toxin effect, pretreatment of human myometrial cells with the tyrosine kinase inhibitor genestein (25 and 50 µM) for 2 h also partially inhibited (28-40%) the stimulation of RGS2 mRNA expression by 100 nM OT (Fig. 7).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Inhibition of OT-stimulated RGS2 mRNA expression by pertussis toxin (PTx) and genestein (Gen), a tyrosine kinase inhibitor. The myometrial cells were pretreated with either pertussis toxin for 18 h or with genestein for 30 min before treatment with OT for 2 h.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The RGS2 mRNA expression in human myometrial cells in primary culture was elevated in the presence of 10% FBS, but it was substantially diminished after 18 h of serum deprivation. Subsequent treatment of cells with OT caused a marked increase in RGS2 mRNA levels after 1 or 2 h, with a rise that was obvious after 30 min. Near-maximal effects were obtained with 1 nM OT, which is near the EC50 value for OT stimulation of intracellular Ca2+ and inositol phosphate elevation in human myometrial cells in primary culture (25). These findings indicate that the effects of OT on RGS2 mRNA are physiologically relevant. The effects of OT were ligand specific, since the OT antagonist blocked the rise in RGS2 mRNA levels.

There is still relatively little known about what regulates different RGS proteins. Based on the limited data available, the signal pathways involved in RGS2 mRNA expression appear to depend on cellular context. In human neuroblastoma SH-SY5Y cells, RGS2 mRNA levels are increased by activation of muscarinic receptors and a PKC-dependent mechanism (26). The RGS2 expression was not affected by increases in either intracellular Ca2+ or cAMP concentrations. PKC also appears to mediate RGS2 mRNA increases by ANG II in vascular smooth muscle cells (11). Tyrosine kinase inhibition and Ca2+ deprivation did not affect this increase in RGS2 mRNA concentration. A greater increase was seen in levels of RGS1 mRNA in human B lymphocytes in response to a PKC activator than to a Ca2+ ionophore (ionomycin), whereas the opposite was true for RGS2 mRNA synthesis in blood mononuclear cells (13). Elevation of intracellular cAMP induces RGS2 expression in cultured rat osteoblasts (19) and PC-12 cells (22). Thus, depending on the cell type, RGS2 expression can be induced exclusively by PKC, intracellular Ca2+, or cAMP. Each one of these pathways mediates the actions of G protein-coupled receptors and likely contributes to feedback regulation of the G proteins involved.

As we have shown in our present studies, there are multiple pathways that lead to increased RGS2 mRNA levels in human myometrial cells (Fig. 8). The primary signal that mediates the effects of OT appears to be linked to PLC-mediated stimulation of intracellular Ca2+ concentrations. OT also activates PKC via PLC-stimulated increases in diacylglycerol concentration. PKC likely catalyzes the phosphorylation of specific proteins that are involved in increased RGS2 mRNA levels. Partial inhibition of the effects of OT on RGS2 mRNA expression by pertussis toxin indicates that Gi/o plays a role in signaling increases in RGS2 mRNA levels. In view of the similar degree of inhibition caused by pertussis toxin and genestein, we speculate that Gbeta gamma linked to tyrosine kinase activation mediates a portion of the OT effect on RGS2 mRNA expression. This type of transactivation by Gbeta gamma has been described in other G protein-coupled receptor systems (16, 17). Increases in intracellular cAMP concentrations also caused a rise in RGS2 mRNA levels, presumably independent of the OT receptor, which is not coupled to Gs. cAMP produces relaxation of smooth muscle by activation of cAMP-dependent protein kinase A, which interferes with several processes involved in smooth muscle contraction (3, 30). We speculate that there is the potential for heterologous regulation of OT action by agents that generate intracellular cAMP. The resulting increase in RGS2 expression might attenuate the responses to OT or other uterotonins acting via G proteins. Agents that increase myometrial cell cAMP levels include beta -adrenergic drugs (15), relaxin (18), corticotropin-releasing hormone (10), calcitonin gene-related peptide (1), adrenomedullin (1), and parathyroid hormone-related peptide (29), among others.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8.   Summary of multiple pathways involved in upregulating RGS2 mRNA expression in human myometrial cells in primary culture. The primary signal mediating the effects of OT was an elevation in intracellular Ca2+ concentration. PKC activation appeared to be secondary. Independent of the effects of OT, forskolin (which elevates intracellular cAMP concentrations) also increased RGS2 mRNA expression. About one-half of the OT effect is mediated by Gi/o and tyrosine kinase activity, as reflected by inhibition by pertussis toxin and genestein, respectively. Tyrosine kinase activation has been shown in other systems to be coupled to Gi activation, presumably through Gbeta gamma interactions. OTR, OT receptor; PLC, phospholipase C; DAG, diacylglycerol; PKA, protein kinase A; InsP3, inositol trisphosphate.

The role of RGS2 in myometrial cell function remains to be established. It has been tacitly assumed that RGS2 functions as a GTPase. Thus an increase in RGS2 protein levels after an initial stimulation might be expected to attenuate subsequent signaling. In support of this possibility, Phaneuf and coworkers (23) found that exposing cultured human myometrial cells to OT for a prolonged period caused desensitization of the response to OT. There was a concomitant 90% reduction in the number of high-affinity receptor binding sites. Immunoblot and flow cytometry data indicated, however, that the total amount of OT receptor protein on the cell membrane surface was unaffected by OT treatment. These findings suggest that desensitization could occur at the G protein level (possibly involving RGS2) rather than at the receptor level.

Recent studies have indicated that RGS2 phosphorylation, along with RGS2 protein synthesis, may also be an important means of regulating GTPase activity. Phosphorylation of RGS2 by PKC in vitro diminished RGS2 activity stoichiometrically (28). There is evidence that RGS2 also has functions that are not associated with GTPase activity. RGS2 inhibits cAMP production by directly inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons (4). Although a clear understanding of the role in RGS2 mRNA in human myometrial cells remains to be established, the profound changes in RGS2 mRNA concentration elicited by OT suggest that RGS2 serves an important regulatory function with respect to OT action in these cells. Upregulation of RGS2 mRNA has also been demonstrated in vivo, in ovarian granulosa cells after human chorionic gonadotropin treatment (27). Because the myometrium undergoes marked changes in sensitivity to OT during pregnancy (6, 7), future studies are needed to elucidate the importance of RGS2 in the responsiveness of the myometrium to OT in vivo.


    ACKNOWLEDGEMENTS

We thank Dr. Garland D. Anderson and the Department of Obstetrics and Gynecology for supporting these studies.


    FOOTNOTES

Current address for C. O. Echetebu: Dept. of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641.

Address for reprint requests and other correspondence: M. S. Soloff, Dept. of Obstetrics & Gynecology, The Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-1062 (E-mail: msoloff{at}utmb.edu).

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. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00437.2001

Received 22 September 2001; accepted in final form 2 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Casey, ML, Smith J, Alsabrook G, and MacDonald PC. Activation of adenylyl cyclase in human myometrial smooth muscle cells by neuropeptides. J Clin Endocrinol Metab 82: 3087-3092, 1997[Abstract/Free Full Text].

2.   Chen, C, Zheng B, Han J, and Lin SC. Characterization of a novel mammalian RGS protein that binds to Galpha proteins and inhibits pheromone signaling in yeast. J Biol Chem 272: 8679-8685, 1997[Abstract/Free Full Text].

3.   De Lanerolle, P, Nishikawa M, Yost DA, and Adelstein RS. Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science 223: 1415-1417, 1984[ISI][Medline].

4.   Dessauer, CW, Srikumar D, Chen J, Yuen J, Yilma S, Dennis JC, Morrison EE, Vodyanoy V, and Kehrl JH. RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III. Nature 409: 1051-1055, 2001[ISI][Medline].

5.   Echetebu, CO, Ali M, Izban MG, Mackay L, and Garfield RE. Localization of regulatory protein binding sites in the proximal region of human myometrial connexin 43 gene. Mol Hum Reprod 5: 757-766, 1999[Abstract/Free Full Text].

6.   Fuchs, AR, Fuchs F, Husslein P, Soloff MS, and Fernstrom MJ. Oxytocin receptors and human parturition: a dual role for oxytocin in the initiation of labor. Science 215: 1396-1398, 1982[ISI][Medline].

7.   Fuchs, AR, Periyasamy S, Alexandrova M, and Soloff MS. Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium: effects of ovarian steroids. Endocrinology 113: 742-749, 1983[Abstract].

8.   Gold, SJ, Ni YG, Dohlman HG, and Nestler EJ. Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain. J Neurosci 17: 8024-8037, 1997[Abstract/Free Full Text].

9.   Grammatopoulos, DK, and Hillhouse EW. Activation of protein kinase C by oxytocin inhibits the biological activity of the human myometrial corticotropin-releasing hormone receptor at term. Endocrinology 140: 585-594, 1999[Abstract/Free Full Text].

10.   Grammatopoulos, DK, and Hillhouse EW. Role of corticotropin-releasing hormone in onset of labour. Lancet 354: 1546-1549, 1999[ISI][Medline].

11.   Grant, SL, Lassegue B, Griendling KK, Ushio-Fukai M, Lyons PR, and Alexander RW. Specific regulation of RGS2 messenger RNA by angiotensin II in cultured vascular smooth muscle cells. Mol Pharmacol 57: 460-467, 2000[Abstract/Free Full Text].

12.   Hepler, JR. Emerging roles for RGS proteins in cell signalling. Trends Pharmacol Sci 20: 376-382, 1999[ISI][Medline].

13.   Heximer, SP, Cristillo AD, and Forsdyke DR. Comparison of mRNA expression of two regulators of G-protein signaling, RGS1/BL34/1R20 and RGS2/G0S8, in cultured human blood mononuclear cells. DNA Cell Biol 16: 589-598, 1997[ISI][Medline].

14.   Ingi, T, Krumins AM, Chidiac P, Brothers GM, Chung S, Snow BE, Barnes CA, Lanahan AA, Siderovski DP, Ross EM, Gilman AG, and Worley PF. Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signaling and neuronal plasticity. J Neurosci 18: 7178-7188, 1998[Abstract].

15.   Lecrivain, JL, Cohen-Tannoudji J, Robin MT, Coudouel N, Legrand C, and Maltier JP. Molecular mechanisms of adenylyl cyclase desensitization in pregnant rat myometrium following in vivo administration of the beta -adrenergic agonist, isoproterenol. Biol Reprod 59: 45-52, 1998[Abstract/Free Full Text].

16.   Leopoldt, D, Hanck T, Exner T, Maier U, Wetzker R, and Nürnberg B. Gbeta gamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit. J Biol Chem 273: 7024-7029, 1998[Abstract/Free Full Text].

17.   Luttrell, LM, Della Rocca GJ, van Biesen T, Luttrell DK, and Lefkowitz RJ. Gbeta gamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation. J Biol Chem 272: 4637-4644, 1997[Abstract/Free Full Text].

18.   Meera, P, Anwer K, Monga M, Oberti C, Stefani E, Toro L, and Sanborn BM. Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A. Am J Physiol Cell Physiol 269: C312-C317, 1995[Abstract/Free Full Text].

19.   Miles, RR, Sluka JP, Santerre RF, Hale LV, Bloem L, Boguslawski G, Thirunavukkarasu K, Hock JM, and Onyia JE. Dynamic regulation of RGS2 in bone: potential new insights into parathyroid hormone signaling mechanisms. Endocrinology 141: 28-36, 2000[Abstract/Free Full Text].

20.   Molnar, M, Rigo J, Jr, Romero R, and Hertelendy F. Oxytocin activates mitogen-activated protein kinase and up-regulates cyclooxygenase-2 and prostaglandin production in human myometrial cells. Am J Obstet Gynecol 181: 42-49, 1999[ISI][Medline].

21.   Morrison, JJ, Dearn SR, Smith SK, and Ahmed A. Activation of protein kinase C is required for oxytocin-induced contractility in human pregnant myometrium. Hum Reprod 11: 2285-2290, 1996[Abstract].

22.   Pepperl, DJ, Shah-Basu S, VanLeeuwen D, Granneman JG, and MacKenzie RG. Regulation of RGS mRNAs by cAMP in PC12 cells. Biochem Biophys Res Commun 243: 52-55, 1998[ISI][Medline].

23.   Phaneuf, S, Asboth G, Carrasco MP, Linares BR, Kimura T, Harris A, and Lopez Bernal A. Desensitization of oxytocin receptors in human myometrium. Hum Reprod 4: 625-633, 1998[Abstract].

24.   Phaneuf, S, Carrasco MP, Europe-Finner GN, Hamilton CH, and Lopez Bernal A. Multiple G proteins and phospholipase C isoforms in human myometrial cells: implication for oxytocin action. J Clin Endocrinol Metab 81: 2098-2103, 1996[Abstract].

25.   Phaneuf, S, Europe-Finner GN, Varney M, MacKenzie IZ, Watson SP, and Lopez Bernal A. Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins. J Endocrinol 136: 497-509, 1993[Abstract].

26.   Song, L, De Sarno P, and Jope RS. Muscarinic receptor stimulation increases regulators of G-protein signaling 2 mRNA levels through a protein kinase C-dependent mechanism. J Biol Chem 274: 29689-29693, 1999[Abstract/Free Full Text].

27.   Ujioka, T, Russell DL, Okamura H, Richards JS, and Espey LL. Expression of regulator of G-protein signaling protein-2 gene in the rat ovary at the time of ovulation. Biol Reprod 63: 1513-1517, 2000[Abstract/Free Full Text].

28.   Waldo, GL, Hollinger S, Hepler JR, and Harden TK. Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling. J Biol Chem 276: 5438-5444, 2001[Abstract/Free Full Text].

29.   Williams, ED, Leaver DD, Danks JA, Moseley JM, and Martin TJ. Effect of parathyroid hormone-related protein (PTHrP) on the contractility of the myometrium and localization of PTHrP in the uterus of pregnant rats. J Reprod Fertil 102: 209-214, 1994[Abstract].

30.   Yue, C, Dodge KL, Weber G, and Sanborn BM. Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta 3 stimulation by Galpha q. J Biol Chem 273: 18023-18027, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 282(3):E580-E584
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society