G Protein Preferences for Dopamine D2 Inhibition of Prolactin Secretion and DNA Synthesis in GH4 Pituitary Cells

Paul R. Albert

Ottawa Health Research Institute, Department of Neuroscience, University of Ottawa, Ottawa, Canada K1H-8M5

Address all correspondence and requests for reprints to: Dr. Paul R. Albert, Ottawa Health Research Institute, Department of Neuroscience, University of Ottawa, 451 Smyth Road, Ottawa, Canada K1H-8M5. E-mail: palbert{at}uottawa.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dopamine is the primary inhibitory regulator of lactotroph proliferation and prolactin (PRL) secretion in vivo, acting via dopamine D2 receptors (short D2S and long D2L forms). In GH4C1 pituitary cells transfected with D2S or D2L receptor cDNA, dopamine inhibits PRL secretion and DNA synthesis. These actions were blocked by pertussis toxin, implicating Gi/Go proteins. To address roles of specific Gi/Go4 proteins in these actions a series of GH4C1 cell lines specifically depleted of individual G{alpha} subunits was examined. D2S-mediated inhibition of BayK8644-stimulated PRL secretion was primarily dependent on Go over Gi, as observed for BayK8644-induced calcium influx. By contrast, inhibitory coupling of the D2S receptor to TRH-induced PRL secretion was partially impaired by depletion of any single G protein, but especially Gi3. Inhibitory coupling of D2L receptors to PRL secretion required Go, but not Gi2, muscarinic receptor coupling was resistant to depletion of any Gi/Go protein, whereas the 5-HT1A and somatostatin receptors required Gi2 or Gi3 for coupling. The various receptors also demonstrated distinct G protein requirements for inhibition of DNA synthesis: depletion of any Gi/Go subunit completely uncoupled the D2S receptor, the D2L receptor was uncoupled by depletion of Gi2, and muscarinic and somatostatin receptors were resistant to depletion of Gi2 only. These results demonstrate distinct receptor-G protein preferences for inhibition of TRH-induced PRL secretion and DNA synthesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROLACTIN (PRL) SECRETION IS regulated primarily by the hypothalamic releasing hormones TRH (stimulatory) and dopamine (DA; inhibitory) (1, 2). TRH stimulates PRL secretion via Gq-mediated activation of phosphatidylinositol turnover (3, 4). DA acts via D2 receptors that couple to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit PRL secretion in lactotrophs (5, 6). Two subtypes of D2 receptors (D2S-short and D2L-long) are generated by alternate splicing of 29 amino acids and display similar receptor pharmacology and coupling properties (7). DA D2 agonists are used clinically to treat pituitary adenomas, suggesting that the D2 receptor can inhibit the growth of pituitary cells in man (8). Furthermore, mice lacking the D2 receptor gene hypersecrete PRL and display hypertrophic pituitaries that form adenomas with age, indicating that the D2 receptor restrains the growth and transformation of lactotrophs in vivo (9, 10). Conversely, knockout of the DA transporter gene, which results in a surfeit of DA release, leads to hypotrophic pituitaries and arrested lactotroph development (11).

GH4C1 cells are a clonal strain of rat pituitary cells in which PRL secretion is stimulated by TRH and is inhibited by somatostatin and carbachol via Gi/Go coupled muscarinic M4 and somatostatin (SSTR2) receptors (3, 12). Like somatostatin and carbachol, DA inhibited both TRH- and vasoactive intestinal polypeptide (VIP)-stimulated PRL secretion and reduced DNA synthesis in D2-transfected GH4C1 cells (13, 14, 15, 16, 17). These receptors initiate several inhibitory signaling pathways, including decreased cAMP levels (basal and hormone stimulated), inhibition of MAPK (18, 19), inhibition of L-type calcium channels, and activation of potassium channels leading to membrane hyperpolarization (20, 21, 22). These actions of DA, carbachol, and somatostatin are completely abolished by pretreatment with PTX (3), which inactivates all Gi/Go proteins. Several studies have emphasized that G protein specificity depends on the receptor subtype and the effector addressed, and is most prominent when observed in intact cells rather than by reconstitution in vitro (23, 24). However, in only a few cases has the G protein specificity of downstream biological responses, such as cell proliferation, transformation, or degranulation, been identified (25, 26). Thus, specific G proteins may be necessary for some or all responses of the D2 receptor.

The present study addresses whether receptor-G protein specificity, which has been observed for specific effectors (24, 27), extends to complex biological responses such as inhibition of PRL secretion or DNA synthesis. To identify the G protein specificity of multiple receptors for these responses, we used a series of GH4C1 cell lines that stably express full-length antisense cDNAs to G{alpha}o, G{alpha}i1, G{alpha}i2, or G{alpha}i3 (28, 29). Individual G proteins were specifically depleted by over 75%, with no depletion of other G{alpha} or Gß proteins detected by Western blot analysis. Furthermore, the inhibitory receptors examined (transfected D2S, D2L, or 5-HT1A; endogenous muscarinic and somatostatin receptors) mediated one or more functional responses that were equivalent in antisense clones and wild-type cells not transfected with antisense G{alpha} cDNA. For example, in the antisense G{alpha}i clones, inhibition of BayK8644-induced calcium channel activation was not different from that in controls; in the antisense G{alpha}o clones, inhibition of VIP-induced cAMP accumulation was not different from that in controls. These clones were examined for receptor-mediated inhibition of basal and TRH-stimulated PRL secretion and DNA synthesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G Protein Specificity of D2-Induced Inhibition of PRL Secretion
PRL secretion in GH4 clones was measured within 45 min, as this represents mainly the release of stored, rather than newly synthesized, PRL (30, 31). In multiple experiments the average basal PRL secretion in antisense clones was not significantly different from that in wild-type cells (Table 1Go). TRH induced between 2- and 3-fold stimulation of basal PRL secretion, on the average, in both wild-type and antisense clones (Table 1Go). Thus, depletion of individual G{alpha}i/G{alpha}o did not impair basal or TRH-induced secretion. This is consistent with the lack of effect of PTX-mediated inactivation of G{alpha}i/G{alpha}o proteins on basal or TRH-induced PRL secretion in these cells (32).


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Table 1. Comparison of Basal and TRH-Stimulated PRL Secretion in Wild-Type and Antisense G{alpha} GH4 Clones

 
The roles of specific G proteins in inhibition of TRH- vs. BayK8644-induced PRL secretion were compared (Fig. 1Go). In D2S-expressing GH4ZR7 cells (Fig. 1Go), DA did not affect basal PRL secretion, but inhibited TRH-induced secretion by 60%, as shown previously (13). BayK8644 induced a 2.5-fold stimulation of PRL secretion, and this effect was blocked by 80% by D2S receptor activation. As predicted from previous data on BayK8644-induced calcium influx, Go, but not Gi1 or Gi2, was required for DA-mediated inhibition of BAY; Gi3 depletion also partially blocked this response. By contrast, DA-mediated inhibition of TRH-induced PRL secretion was inhibited by depletion of Go or Gi2 and was poorly inhibited by Gi3 depletion; Gi1 was less effective. For the D2L receptor in GH4D2L cells (Fig. 2Go), depletion of Go entirely blocked the receptor action, as observed for D2S (Fig. 1Go), but depletion of Gi2 was ineffective. Depletion of Go and Gi2 reduced carbachol and somatostatin action, although Gi2 depletion preferentially inhibited somatostatin action. These examples illustrate the different G protein specificities of various receptors in individual clones.



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Figure 1. Distinct G Protein Specificity for D2S-Mediated Inhibition of TRH- or BayK8644-Enhanced PRL Secretion

GH4ZR7 cells (stably expressing the DA D2S receptor) or derivative clones expressing antisense RNA for G{alpha}o (GoZR7), G{alpha}i1 (G11ZR7), G{alpha}i2 (Gi2ZR7), or G{alpha}i3 (Gi3ZR7) were assayed for PRL secretion as described in Materials and Methods. The concentrations of experimental compounds were: TRH, 1 µM; DA, 1 µM; and BayK8644 (BAY), 1 µM. Data are presented as the mean ± SE of triplicate samples, and the percent inhibition by DA of TRH- and BayK8644-induced secretion is shown numerically above for each cell line.

 


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Figure 2. G Protein Specificity of D2L, Muscarinic- and Somatostatin Receptor-Mediated Inhibition of TRH-Induced PRL Secretion

GH4D2L cells (stably expressing the DA D2L receptor) or derivative clones expressing antisense RNA for G{alpha}o (GoD2L) or G{alpha}i2 (Gi2D2L) were assayed for PRL secretion as described in Materials and Methods. The compounds tested were TRH (1 µM), DA (1 µM), somatostatin (SS; 250 nM), and carbachol (M; 1 µM). Data are presented as the mean ± SE of triplicate samples, and the percent inhibition by DA/somatostatin/carbachol of TRH-induced secretion is shown numerically above for each cell line.

 
Several experiments, shown in Figs. 1Go and 2Go, were performed on multiple clones, and the average percent inhibition (see Materials and Methods) of TRH-induced PRL secretion by DA D2S, DA D2L, 5-HT1A receptor, muscarinic M4, and SSTR receptor activation in the various wild-type and antisense G{alpha} cell lines is presented (Fig. 3Go). Each receptor inhibited TRH-induced PRL secretion with an order of efficacy similar to that observed for receptor coupling to inhibition of adenylyl cyclase in these cells (28, 29). For the D2S receptor, depletion of any individual Gi protein impaired receptor action, and depletion Gi3 had the largest effect. By contrast, for the D2L receptor, loss of Go almost completely impaired inhibition of TRH-induced secretion, whereas Gi2 depletion had no significant effect. In contrast, there was no significant overall effect of depletion of any individual Gi/Go subunit on muscarinic inhibition, whereas the actions of 5-HT and somatostatin were dependent on Gi2 or Gi3. These data show that the different G protein specificities of various receptors in independent cell lines are dependent on the receptor subtype.



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Figure 3. Summary of G Protein Specificity for Inhibition of TRH-Induced PRL Secretion by Multiple Receptors

Data from multiple experiments (3–25 for each ligand in each cell line), as described in Figs. 1Go and 2Go, are presented as the mean ± SE of the percent inhibition of TRH-induced PRL secretion by each ligand calculated as described in Materials and Methods. For each receptor the cell lines tested were wild type (WT; not transfected with antisense G{alpha}) or cells transfected with antisense to G{alpha}o, G{alpha}i1, G{alpha}i2, or G{alpha}i3 (Go, Gi1, Gi2, and Gi3, respectively). *, P < 0.05; ***, P < 0.001 (compared with WT cells by one-way ANOVA by Dunnett’s comparison test). nd, Not determined. All lanes were significantly different from control (untreated) cells.

 
G Protein Specificity for DA-Induced Inhibition of DNA Synthesis
In GH4ZR7 cells, one study found that DA D2S receptor activation inhibited DNA synthesis by 30%, but was not blocked by PTX (33), whereas in another study PTX did block the response (17). Two protocols were compared for testing DA inhibition of [3H]thymidine incorporation in GH4ZR7 cells as a measure of DNA synthesis (Fig. 4AGo): extraction with trichloroacetic acid, then NaOH/sodium dodecyl sulfate (34) or perchloric acid extraction (33). In both cases DA inhibited DNA synthesis in a concentration-dependent manner. Two PTX treatment paradigms were used: simultaneous addition of PTX with DA or 24-h pretreatment with PTX before DA addition, as reported by Senogles (33). In the latter case, DA-induced inhibition of DNA synthesis was not blocked by PTX, presumably due to replacement of PTX-inactivated G proteins by newly synthesized proteins during the subsequent 20-h assay period. However, with coaddition of PTX and DA a complete blockade of DA action was obtained, indicating that PTX blocked D2-induced inhibition of DNA synthesis within 4 h of addition. Furthermore, the action of PTX to block DA-induced inhibition of DNA synthesis was concentration dependent (Fig. 4BGo). The average EC50 value was 1.3 ± 0.4 ng/ml in three independent experiments, similar to the EC50 value of 0.3 ng/ml for PTX-induced blockade of somatostatin-mediated inhibition of PRL secretion in GH4 cells (35). Thus, DA-mediated inhibition of DNA synthesis in GH4ZR7 cells is blocked by PTX treatment.



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Figure 4. Pertussis Toxin-Sensitive Inhibition of DNA Synthesis by D2S Receptor Activation

A, Blockade by PTX depends on the protocol used. DNA synthesis was measured as the percentage of [3H]thymidine incorporation in control (untreated GH4ZR7 cells). Data are expressed as the mean ± SE of triplicate samples. Cells were treated with DA at the indicated concentration. Two extraction protocols (see Materials and Methods) were used to determine [3H]thymidine incorporation in GH4ZR7 cells: extraction with trichloroacetic acid, followed by recovery in NaOH/sodium dodecyl sulfate (34 ) or extraction with perchloric acid (33 ). In both cases DA inhibited DNA synthesis to a similar extent. Two PTX (50 ng/ml) treatment paradigms were used: simultaneous addition with DA (10 µM; DA+PTX) or 24-h pretreatment with PTX before DA addition (PTX/DA) as reported by Senogles (33 ). In the latter case DA inhibited DNA synthesis, presumably due to replacement of PTX-inactivated G proteins by new protein synthesis during the subsequent 20-h assay period. However, with simultaneous PTX+DA addition, a complete and consistent blockade of DA action was obtained, suggesting that PTX is active within 4 h of addition. B, The concentration dependence of PTX action to inhibit D2S receptor-mediated inhibition of DNA synthesis in GH4ZR7 cells was determined. The indicated concentrations of PTX were added in the presence of 1 µM DA, and [3H]thymidine incorporation was determined as a percentage of the control (mean ± SE of triplicate samples). In this experiment, an EC50 value of 2.3 ng/ml was obtained by nonlinear regression analysis using the PRISM 3.0 program, and the corresponding curve is plotted.

 
In multiple experiments in both GH4ZR7 and GH4D2L cells, DA induced a 25–30% inhibition of DNA synthesis (Fig. 5Go). These responses were blocked by PTX, which had no effect on its own. Furthermore, activation of muscarinic or somatostatin receptors also inhibited DNA synthesis. To determine which G proteins mediated the response, the panel of antisense G{alpha} cell lines was examined for the actions of DA, carbachol, and somatostatin to inhibit DNA synthesis. For the D2S receptor, depletion of any single G protein was sufficient to block DA-induced inhibition of DNA synthesis. Similarly, depletion of Go or Gi2 blocked D2L-induced inhibition of DNA synthesis. For the muscarinic and somatostatin receptors, inhibition of DNA synthesis was resistant to depletion of Gi2. Thus, multiple G proteins participate in receptormediated inhibition of DNA synthesis.



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Figure 5. G Protein Specificity for Inhibition of DNA Synthesis by Multiple Receptors

Data from multiple experiments (two to nine experiments for each ligand in each cell line) are the mean ± SE of [3H]thymidine incorporation presented as the percent inhibition compared with that in control untreated cells. Addition of PTX (50 ng/ml) with DA is shown (AG/PTX). For each receptor, wild-type cells (W. T.; not transfected with antisense G{alpha}) or cells transfected with antisense to G{alpha}o, G{alpha}i1, G{alpha}i2, or G{alpha}i3 (AS-Go, Gi1, Gi2, or Gi3, respectively) were tested. *, P < 0.05; ***, P < 0.001 (compared with control, untreated cells). nd, Not determined. Points that were not different from control displayed significant inhibition compared with wild type.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Role of Go in Inhibition of BayK8644 Action
BayK8644 directly activates L-type calcium channels to induce calcium influx, which, in turn, triggers PRL secretion (36, 37). Our results indicate a crucial role for Go in D2S coupling to inhibition of BayK8466-induced PRL secretion, but not to inhibit TRH-induced secretion. The crucial role of Go in inhibition of BayK8644-induced PRL secretion is consistent with the role of Go in D2S-, D2L-, 5-HT1A-, muscarinic-, and somatostatin-induced reduction of BayK8644-stimulated calcium influx in these cells (28, 29). It remains unclear exactly how Go mediates inhibition of calcium influx via L-type channels. Receptor-specific combinations of G{alpha}-Gß{gamma} subunits (23, 24) may directly modulate heteromeric L-type channels containing Gß{gamma}-sensitive channel {alpha}1A-subunits (38). Although G{alpha}i3 does not couple to calcium channels in these cells (29, 39, 40, 41), depletion of G{alpha}i3 also partially impaired D2S-induced inhibition of BayK8644-induced secretion. Gi3 has been implicated in D2-mediated regulation of potassium channels (39, 40, 41), and this pathway leads to membrane hyperpolarization that could also participate indirectly in DA action to close voltage-dependent calcium channels.

G Protein Specificity for Inhibition of TRH-Induced Secretion
In contrast to BayK8644-stimulated secretion, TRH-mediated secretion involves the recruitment of several signaling pathways. TRH couples to Gq to activate phospholipase C, generating the dual second messengers, inositol trisphosphate and diacylglycerol (42, 43). TRH-induced PRL release occurs in two phases: an initial burst phase that requires inositol trisphosphate-mediated release of calcium stores, and a sustained phase that involves calcium influx in part through protein kinase C-mediated activation of voltage-dependent calcium channels (43, 44, 45, 46). In addition, TRH activates p42/44-MAPK in GH4 cells within minutes (47, 48, 49), but the role of this pathway in TRH-induced secretion has yet to be addressed. The requirement of several G proteins for inhibition of TRH-induced secretion probably reflects the diversity of pathways that may be involved in TRH-mediated secretion. However, depletion of G{alpha}i3 induced the greatest impairment of inhibition for several receptors, including D2S, somatostatin, and 5-HT1A. This is consistent with blockade of D2 inhibition of TRH-induced secretion by injection of anti-G{alpha}i3 antibodies (50, 51). The general importance of Gi3 for these multiple receptors is consistent with a role for Gi3 in a late common step controlling vesicle fusion (52). Alternately, we recently identified Go and Gi3 as crucial for D2S-mediated inhibition of MAPK in GH4ZR7 cells (53), suggesting a MAPK-linked mechanism for Gi3induced inhibition of PRL secretion. As depletion of any individual G protein, including Gi3, did not completely block inhibition of PRL secretion, compensatory signaling by other Gi/Go proteins is likely. In contrast, in brains from mice deficient in the Go gene, D2-induced G protein coupling was largely attenuated (54). The present data provide the first evidence that multiple Gi/Go proteins are required to inhibit TRH-induced PRL secretion, although for several receptors, Gi3 appears to be the most important.

D2 Receptor Specificity of G Protein Coupling
Different receptors displayed distinct G protein preferences for inhibition of TRH-induced secretion. Importantly, DA D2S and D2L receptors, which differ by only 29 amino acids (7), had different sensitivities to depletion of Go and Gi2. The D2L receptor was insensitive to depletion of Gi2, but relied on Go, whereas the D2S response was not significantly altered by Go depletion, but was sensitive to reductions in Gi1, Gi2, and Gi3. Differences in G protein selectivity of D2S and D2L receptors have been reported previously. Gi2 was required for D2L-mediated inhibition of forskolin-stimulated adenylyl cyclase (55, 56), but not for D2S-mediated inhibition of Gs-stimulated adenylyl cyclase (28, 57). Mutagenesis of the 29-amino acid splice domain reduced the D2L requirement for Gi2, suggesting that this domain influences the G protein specificity of the D2 receptor (58). The D2L receptor was the only receptor tested in which Go played an important role in G protein coupling to inhibition of PRL secretion. This suggests that, as observed in brain, Go may play a particularly important role in D2L-mediated responses (54). These results indicate that differential G protein specificity of the D2 receptors extends to regulation of biological responses such as inhibition of PRL secretion.

G Protein Specificity for Inhibition of DNA Synthesis
Gi/Go protein-mediated inhibition of DNA synthesis in pituitary cells is incompletely understood but appears to involve multiple signaling pathways. For D2induced inhibition of cell proliferation, pathways including activation of protein kinase C{epsilon} (33), inhibition of protein tyrosine phosphatase activation (17), and activation of p38 MAPK or inhibition of p42/44 MAPK activation (48) have been suggested as potential mediators. D2-induced inhibition of DNA synthesis was sensitive to PTX, suggesting that the PTX-insensitive activation of protein kinase C{epsilon} is unlikely to play a major role. For the D2S receptor this response required coactivation of several G proteins, and depletion of any single Gi or Go subtype blocked inhibition of DNA synthesis. In GH4ZR7 cells, Gi2 did not appear to participate in D2S inhibition of TRH-induced MAPK, but Go and Gi3 did (53). The D2S receptor may use Go and Gi3 to inhibit MAPK, but other G proteins appear to target different pathways and reduce cell proliferation. Interestingly, inhibition of DNA synthesis by somatostatin and muscarinic receptors was retained in cells depleted of Gi2. Depletion of Gi2 blocked inhibition of basal and VIP-stimulated cAMP formation by these receptors (28, 29), suggesting that cAMP inhibition is dispensable for inhibition of DNA synthesis. In contrast to secretion, inhibition of [3H]thymidine incorporation was completely, rather than partially, blocked upon depletion of individual G proteins. This indicates that D2S-induced inhibition of [3H]thymidine incorporation requires coordinate mediation by several G proteins, whereas inhibition of specific effectors (L-type channels, adenylyl cyclase) or secretion requires fewer G proteins.

G Protein Specificity for Biological Responses
Together these results demonstrate distinct receptor-G protein preferences for biological responses, including inhibition of TRH-induced PRL secretion and DNA synthesis. These antisense studies represent a relatively nonperturbing approach that differs from pharmacological or dominant-negative inhibition of downstream effectors. Depletion of individual G protein subunits is proximal to the receptor and did not have global effects on basal proliferation or secretion, yet revealed specific roles in receptor action. Preliminary mapping of receptor-G protein specificity of effectors and biological responses indicates that Gi3-mediated signaling is most important for inhibition of PRL secretion by several receptors. In addition, for certain receptors, inhibition of DNA synthesis was preserved in cells where inhibition of cAMP is lost.

Compared with other receptors, the DA-D2S receptor recruited a larger variety of G proteins to mediate downstream responses that may reflect stronger activation of several Gi/Go proteins. Affinity purification or immunopurification of receptors has demonstrated that agonist-bound somatostatin receptors remain associated with specific G proteins that do not correspond to their abundance in cell membranes (59, 60, 61, 62, 63). Identification of specific blockers of receptor-G protein interaction may provide novel and highly specific tools to block specific signals of the receptor (64, 65), such as Go-dependent inhibition of L-type calcium channel activation. Inhibition of specific receptor-G protein signaling cascades could selectively block some, but not all, biological responses elicited by a receptor.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
GH4C1 clones were grown in Ham’s F-10 medium and 5% fetal calf serum at 37 C in 5% CO2. Cell lines stably transfected with receptors, including GH4ZR7 (D2S) (13), GH4D2L (D2L) (66), and GH4ZD10 (5-HT1A) (67), were then transfected again with individual antisense G{alpha} constructs and have been characterized by Western blot against G{alpha} and Gß subunits as previously described (28, 29). Stock cultures of antisense clones were periodically selected using 200 µg/ml hygromycin B (Calbiochem, La Jolla, CA), for 7–10 d. Hygromycin was omitted from experimental dishes.

PRL Assay
Cells (106/well) were plated in six-well dishes and fed 24 h before experiments. The cells were rinsed with serum-free Ham’s F-10 medium and 20 mM HEPES (pH 7.2) for 5–10 min at 37 C, then changed to the same medium containing experimental compounds (present in combination) and incubated for 30–60 min, depending on the assay. Previous studies in GH3 cells [from which GH4 cells were subcloned (12)] have shown that TRH-induced release of stored PRL is complete with 30–60 min, whereas release at later times represents primarily release of newly synthesized PRL (30, 31). In all experiments maximal concentrations of agonists were used and diluted 1000-fold from concentrated stock solutions, specifically, TRH, (1 µM), DA (1 µM), serotonin (1 µM), somatostatin-14 (250 nM), and carbachol (1 µM). BayK8644 was used at 1 µM (BAY). This procedure measures primarily the release of previously stored PRL. Levels of rat PRL were quantitated by specific RIA (NIDDK) using [125I]rat PRL (NEN Life Science Products, Boston, MA) as tracer. Antibody dilution was 1:40,000, and the mean EC50 for PRL (RP-5) was 1 ng. Data are presented as the mean ± SEM of triplicate samples assayed in duplicate. The percent inhibition of TRH action by agent X was calculated: (X + TRH-X)/(TRH-C) x 100, where C, X, TRH, and X + TRH refer to the values of PRL in controls or cells treated with ligand X, TRH, or both ligand X and TRH. A similar formula was used to calculate the percent inhibition of BAY action.

DNA Synthesis
Cells (106/well) were plated in six-well (35-mm) dishes 24 h before experiments. The medium was changed to medium containing filter-sterilized 20 mM HEPES (pH 7.0), 0.1% l-ascorbic acid, and experimental compounds and incubated at 37 C in 5% CO2 for 4 h. [3H]Thymidine (1 µCi/well) was added, and the cells were incubated for an additional 16 h. The medium was aspirated, and cells were rinsed with 1 ml PBS, then incubated in 1 ml ice-cold 10% trichloroacetic acid for 30 min at 4 C. The cells were rinsed once with trichloroacetic acid and solubilized in 0.5 ml 0.4M NaOH/1% sodium dodecyl sulfate for scintillation counting in 5 ml cocktail. This extraction procedure (34) extracts counts per minute values at least twice those obtained using the perchloric acid extraction (33). Except where indicated, maximal concentrations of agonists were used and diluted 1000-fold from concentrated stock solutions. Data are presented as the mean ±SEM of triplicate samples normalized to the untreated sample x 100 (i.e. percentage of control) or as indicated.

Statistical Analysis
Data were analyzed using the PRISM 3.0 statistical analysis program by one-way ANOVA, with Dunnett’s posttest to compare with wild type (not transfected with antisense) and Tukey test to compare with control. Significance was set at P < 0.05.


    ACKNOWLEDGMENTS
 
The technical support of Dolores Raquidan is gratefully acknowledged.


    FOOTNOTES
 
This work was supported by the National Cancer Institute, the Canada and Ontario Mental Health Foundation. P.R.A. was a recipient of the Novartis/Canadian Institutes for Health Research Michael Smith Chair in Neuroscience.

Abbreviations: DA, Dopamine; PRL, prolactin; PTX, pertussis toxin; SSTR, somatostatin receptor; VIP, vasoactive intestinal polypeptide.

Received for publication December 7, 2001. Accepted for publication April 10, 2002.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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