Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

₁-Adrenergic receptors (ARs) are heptahelical membrane proteins that mediate some of the actions of norepinephrine and epinephrine. Three subtypes (_1a, _1b, and _1d) of ₁-ARs have been cloned and pharmacologically characterized (Graham et al., 1996), and each is widely expressed in tissues (Scofield et al., 1995). ₁-ARs play important physiological roles in mediating smooth muscle contraction (Graham et al., 1996), epithelial transport (Gesek, 1999), cellular metabolism (Urcelay et al., 1993), and central nervous system functions (Stone et al., 2001). ₁-ARs may also be involved in the etiology of several important diseases, including hypertension (Veglio et al., 2001), cardiac hypertrophy (Knowlton et al., 1993), and benign prostatic hyperplasia (Malloy et al., 1998). Signaling through ₁-ARs is accomplished by activation of a number of different signaling molecules, including phospholipase C (PLC), adenylyl cyclase, and the mitogen-activated protein kinases (MAPKs).

₁-ARs predominantly couple to the phosphoinositide hydrolysis pathway through the guanine nucleotide binding regulatory proteins G_q/11, resulting in phospholipid hydrolysis by PLC and the production of the second messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), which mobilize intracellular Ca²⁺ and activate protein kinase C (PKC), respectively (Exton, 1985; Graham et al., 1996). ₁-ARs can also activate a variety of other signaling molecules, including adenylyl cyclase (Johnson and Minneman, 1986; Perez et al., 1993). There is also evidence that ₁-ARs stimulate the phosphorylation of MAPKs (Michelotti et al., 2000), which are traditionally associated with growth factor receptor signaling. MAPKs belong to a family of serine/threonine kinases that includes the mitogenic ERK1/2 proteins as well as the "stress-related" proteins c-Jun NH₂-terminal kinase and p38 (Lewis et al., 1998). These kinases are points of convergence for cell surface signals (growth factors, neurotransmitters, hormones, and cellular stresses) that regulate cellular growth, division, differentiation, and apoptosis. The mechanisms of MAPK pathway activation by growth factors and cellular stresses are well characterized and have been shown to occur via highly conserved cascades that involve tyrosine phosphorylation and protein-protein association events, ultimately leading to the activation of nuclear transcription factors (Lewis et al., 1998). In the cascade for ERK1/2 activation, members of the Raf family of protein kinases activate MAPK kinases 1/2 (MEK1/2), which in turn activate ERK1/2 (Alessi et al., 1995; Lewis et al., 1998).

The precise mechanisms by which catecholamines act through ₁-ARs to activate MAPK pathways are unclear. In most cell lines studied, ₁-AR stimulation of MAPK pathways requires a PLC-induced increase in cytosolic Ca²⁺ (Romanelli and van de Werve, 1997; Hu et al., 1999). However, in some models, activation of the MAPKs is Ca²⁺-independent (Berts et al., 1999). The dependence of MAPK activation upon PKC activation by ₁-ARs also varies among cell types (Romanelli and van de Werve, 1997; Berts et al., 1999; Hu et al., 1999; Snabaitis et al., 2000). The conflicting reports regarding the roles of Ca²⁺ and PKC in the activation of MAPK pathways by ₁-ARs are likely due to differences in cell phenotypes used in these studies. Thus, the participation of each pathway in cellular events must be verified for each cellular model of _1a-AR expression.

Several reports have shown that ₁-AR subtypes can, in addition to activation of PLC and PKC, increase cAMP accumulation (Johnson and Minneman, 1986; Atkinson and Minneman, 1991; Schwinn et al., 1991; Horie et al., 1995); however, a detailed study of the mechanism of ₁-AR-stimulated cAMP accumulation has not been attempted in the same cells in which Ca²⁺, PKC, and ERK1/2 studies have been carried out. Interestingly, the activation of protein kinase A (PKA) seems to inhibit ERK1/2 phosphorylation (Burgering et al., 1993; Crespo et al., 1995). Because ₁-ARs reportedly produce signals that can both stimulate and inhibit ERK1/2 activation, it is important to determine whether these two pathways interact to regulate ERK1/2 phosphorylation. Thus, we hypothesized that concomitant activation of cAMP pathway blunts the PLC-mediated phosphorylation of ERK1/2. In the present study, we investigated the G protein-coupled signaling pathways that are involved in the activation of ERK1/2 after stimulation of _1a-ARs and the interaction among these pathways in _1a-AR-transfected CHO-K1 cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Phorbol 12-myristate 13-acetate (PMA), BAPTA, bisindolylmaleimide, PD 98059, SQ 22536, and 4-cyano-3-methylisoquinoline (CMQ) were purchased from Calbiochem (San Diego, CA). Fura-2/AM was purchased from Molecular Probes (Eugene, OR). ()-Norepinephrine-HCl, propranolol, isoproterenol, prazosin, forskolin, and Cremophor EL were purchased from Sigma-Aldrich (St. Louis, MO). 5-Methyl-urapidil and BMY-7378 were purchased from Sigma/RBI (Natick, MA). [³H]Prazosin was purchased from PerkinElmer Life Sciences (Boston, MA). [³H]cAMP assay kits were purchased from Diagnostic Products (Los Angeles, CA).

Transfection. The full-length mouse _1a-AR cDNA (1431 base pairs) was subcloned and inserted into the mammalian vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA) for expression. After polymerase chain reaction of the 1431-base pair product using subtype-specific primers (Xiao et al., 1998), the polymerase chain reaction product was transformed into INVF' (Invitrogen) and screened for ampicillin-resistant recombinant plasmids. Ampicillin-resistant colonies were isolated and grown in Luria-Bertani medium containing 50 µg/ml ampicillin, and plasmid DNA was extracted according to the Endofree Plasmid Maxi Protocol (QIAGEN, Valencia, CA). The cloned mouse _1a-AR was subcloned into the modified eukaryotic expression plasmid PMT2'. CHO-K1 cells (kindly provided by Dr. Myron Toews, University of Nebraska Medical Center, Omaha, NE) were stably transfected with mouse _1a-ARs by the LipofectAMINE standard method (Invitrogen). All cells were maintained in a humidified atmosphere at 37°C in Ham's F-12 medium containing 5% fetal bovine serum, 10 U/ml penicillin, 100 µg/ml streptomycin, and 200 µg/ml geneticin sulfate (Invitrogen). _1a-AR expression was verified and characterized using saturation and competition studies with [³H]prazosin, as previously described (Xiao et al., 1998).

Measurement of Intracellular Ca²⁺ Concentration. _1a-AR-transfected and -untransfected CHO-K1 cells were grown to confluence in glass-bottomed culture dishes and were rinsed of growth medium with a 4-morpholinepropanesulfonic acid-buffered solution (solution A) consisting of 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 6 mM glucose, 10 mM Na-3-(N-morpholino)propanesulfonic acid, and 5 mM NaHCO₃. Cells were then incubated for 45 min at 37°C in a loading solution consisting of solution A plus 0.1 mg/ml bovine serum album, 0.02% Cremophor EL, and 2 µM fura-2/AM. Cells were then rinsed twice with warm solution A and allowed to incubate in solution A for an additional 15 min to permit complete hydrolysis of any intact ester linkages on intracellular fura-2/AM. Modified culture dishes containing _1a-AR-transfected CHO-K1 cells were mounted in a thermostatically controlled chamber affixed to the stage of a TE-300 inverted phase-contrast microscope (Nikon, Tokyo, Japan) (Yang et al., 1996). _1a-AR-transfected CHO-K1 cells were incubated in a bathing medium of the following composition: 120 mM NaCl, 5 mM KCl, 0.59 mM KH₂PO₄, 0.6 mM Na₂HPO₄, 20 mM glucose, 2.5 mM CaCl₂, and 10 mM HEPES. Addition of norepinephrine (1 nM-0.1 mM) to culture dishes was accomplished by replacement of the bathing medium with bathing medium containing norepinephrine via modified Pasteur pipettes clamped to the microscope stage. After each response, norepinephrine was removed by replacing the bathing media. In these experiments, there was a 10-min interval between addition of different norepinephrine concentrations to allow for refilling of intracellular Ca²⁺ stores. To determine the conditions necessary to chelate intracellular Ca²⁺ mobilized by norepinephrine, _1a-AR-transfected CHO-K1 cells were preloaded with either 10 or 50 µM of the Ca²⁺ chelator BAPTA for 1 h. Ca²⁺ responses were measured as the fluorescent emission ration of fura-2 alternately excited at 340 and 380 nm (F340/F3380). Changes in intracellular Ca²⁺ were quantified by determining peak fluorescence ratios after norepinephrine treatments.

Western Blotting Experiments. All experiments were performed in confluent monolayers of CHO-K1 cells transfected with _1a-ARs, which were serum starved overnight before the experiments. The monolayers were washed twice with Krebs-Henseleit buffer containing 126 mM NaCl, 5.5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 11.1 mM dextrose, and 0.029 mM Na₂Ca-EDTA, pH 7.4. Norepinephrine treatments were carried out in a humidified incubator at 37°C. After norepinephrine stimulation, the incubation buffer was removed, the cells were lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.2 µM aprotinin, and 10 nM okadaic acid, and centrifuged at 12,000g for 15 min. The pellets were discarded, and the total protein content of the supernatants was determined as described by Bradford (1976). The cell lysates were boiled for 5 min and resolved (5 µg of total protein for ERK1/2) on 4 to 15% SDS-polyacrylamide gradient gels (Bio-Rad, Hercules, CA). The gels were transferred onto nitrocellulose membranes (MSI, Westborough, MA), blocked with 5% bovine serum album for 1 h at room temperature, and incubated overnight with antibodies directed against phospho-ERK1/2 (New England Biolabs, Beverly, MA) at a dilution of 1:1,000. To normalize for protein loading, antibodies recognizing total ERK1/2 (New England Biolabs) were used at a 1:1000 dilution. The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (New England Biolabs) diluted 1:1000 in 1% bovine serum album and 1% dry milk, and visualized by the ECL chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). The densities of the bands (both the 42- and 44-kDa bands for ERK2 and ERK1) were analyzed by densitometry. The densities of the bands for phosphorylated ERK1/2 were divided by band densities for total ERK1/2 to normalize for protein loading and the mean ± S.E.M. of the normalized optical densities were plotted.

Norepinephrine-Stimulated ERK1/2 Phosphorylation. Using antibodies specific for the phosphorylated forms of ERK1/2 in Western blotting, we investigated the kinetics, specificity, second messenger dependence of ERK1/2 pathway phosphorylation, and the inhibitory effect of cAMP on ERK1/2 phosphorylation induced by _1a-AR activation in transfected CHO-K1 cells. The time course of norepinephrine-stimulated ERK1/2 phosphorylation was measured after 2-, 5-, 10-, and 30-min incubation with 10 µM norepinephrine. To obtain norepinephrine concentration-response curves for ERK1/2 phosphorylation, _1a-AR-transfected CHO-K1 cells were stimulated with six different concentrations of norepinephrine ranging from 1 nM to 0.1 mM. To show that norepinephrine-stimulated ERK1/2 phosphorylation was due to _1a-ARs rather than -ARs, _1a-AR-transfected CHO-K1 cells were preincubated with the ₁-AR antagonist prazosin (1 µM), the -AR agonist isoproterenol (1 µM), and the -AR antagonist propranolol (1 µM) for 1 h before treatment with norepinephrine (10 µM). The specificity of norepinephrine-stimulated phosphorylation of ERK1/2 was tested by incubating _1a-AR-transfected CHO-K1 cells with the MEK1/2 inhibitor PD 98059 (50 µM) for 1 h before norepinephrine stimulation. Experiments to study the effects of intracellular Ca²⁺ on norepinephrine-stimulated ERK1/2 phosphorylation were performed in _1a-AR-transfected CHO-K1 cells incubated for 1 h with 50 µM BAPTA before 10 µM norepinephrine stimulation. The involvement of PKC in norepinephrine-stimulated ERK1/2 phosphorylation was investigated in cells preincubated with bisindolylmaleimide I (100 nM) for 1 h before 10 µM norepinephrine stimulation. The effects of adenylyl cyclase and PKA on norepinephrine-induced ERK1/2 phosphorylation were investigated by preincubating the cells with the adenylyl cyclase inhibitor SQ 22536 (50 and 100 µM) or the PKA inhibitor CMQ (300 nM) (Lu et al., 1996) for 1 h before norepinephrine stimulation.

cAMP Assay. _1a-AR-transfected CHO-K1 cells were grown to confluence in 12-well plates. Ham's F-12 media were aspirated and cells were washed twice with 2 ml of HEPES-buffered Krebs' solution consisting of 111 mM NaCl, 5.5 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 1.25 mM NaH₂PO₄, and 11 mM HEPES, 9 mM Na HEPES, 25 mM NaHCO₃, 11.1 mM dextrose, 0.029 mM Na₂Ca EDTA, and 0.5 mM 3-isobutyl-1-methylxanthine, pH 7.4, at 37°C. _1a-AR-transfected CHO-K1 cells were then incubated with 1 µM prazosin, 1 µM propranolol, 50 µM BAPTA, or 100 µM forskolin for 1 h before treatment with 10 µM norepinephrine or 1 µM isoproterenol for 10 min at 37°C. _1a-AR-transfected CHO-K1 cells were then lysed by adding 90% ethanol, and the plates were placed on a rocker at 37°C until the ethanol evaporated. The dried cell lysates was dissolved in Tris buffer (50 mM Tris-HCl and 4 mM EDTA, pH 7.5), and the cAMP concentration was measured using a [³H]cAMP assay kit (Diagnostic Products) according to the manufacturer's instructions.

Statistics. Data are shown as means ± S.E.M. Means were compared by using one-way analysis of variance and the Student-Newman-Keuls test. Differences among groups were considered significantly different if P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of _1a-ARs in CHO-K1 Cells. Cell membranes from _1a-AR-transfected CHO-K1 cells had an _1a-AR density of 1.1 ± 0.2 pmol/mg of protein, with a high affinity (K_d = 0.4 ± 0.1 nM) for [³H]prazosin. The K_i values for the _1a-AR-selective antagonist 5-methylurapidil (0.44 ± 0.06 nM) and for the _1d-AR-selective antagonist BMY-7378 (42.0 ± 0.05 nM) were consistent with affinity values previously reported by our group for the mouse _1a-AR (Xiao et al., 1998). No specific [³H]prazosin binding was detected in membranes isolated from nontransfected CHO-K1 cells (data not shown).

Coupling of _1a-ARs to Mobilization of Intracellular Ca²⁺. To determine whether the mouse _1a-AR was functionally coupled to intracellular Ca²⁺ mobilization in our _1a-AR-transfected CHO-K1 cell model, norepinephrine-induced Ca²⁺ mobilization was measured using the fluorescent Ca²⁺ indicator fura-2. In _1a-AR-transfected CHO-K1 cells, norepinephrine caused a transient increase in the ratio of fura-2 fluorescence (F340/F380) that lasted approximately 1 min before returning to baseline. Norepinephrine stimulated increases in the fluorescence ratio of fura-2 in a concentration-dependent manner (Fig. 1A). The peak Ca²⁺ responses were plotted and a mean concentration-response curve is shown in Fig. 1B. Nonlinear regression analyses gave and EC₅₀ of 122.8 ± 2.9 nM for norepinephrine-stimulated Ca²⁺ mobilization. The ratio of fura-2 fluorescence remained unchanged after norepinephrine stimulation of untransfected CHO-K1 cells (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Concentration-response curve for norepinephrine (NE)-stimulated elevation in intracellular free Ca2+ in 1a-AR-transfected CHO-K1 cells loaded with the fluorescent Ca2+ indicator fura-2. A, Ca2+ responses were measured as the fluorescent emission ratio of fura-2 alternatively excited at 340 and 380 nm (F340/F380). 1a-AR-transfected CHO-K1 cells were treated with different concentrations of NE. NE was removed after the return of the response to baseline. Arrows represent the addition of NE to the cells. Data are typical of three experiments. B, mean norepinephrine-induced Ca2+ mobilization (n = 3) was plotted as a percentage of maximal response versus NE concentration.

Norepinephrine Stimulation of ERK1/2 Activation in _1a-AR-Transfected CHO-K1 cells. We next investigated whether the activation of _1a-ARs in our _1a-AR-transfected cell system stimulated the phosphorylation of ERK1/2. Norepinephrine (10 µM) stimulated the phosphorylation of ERK1/2 in a time-dependent manner (Fig. 2, A and B) with maximal stimulation at 10 min. Norepinephrine also stimulated the phosphorylation of ERK1/2 in a concentration-dependent manner (Fig. 2C). The EC₅₀ (220.6 ± 5.3 nM) for norepinephrine-stimulated ERK1/2 phosphorylation was derived from concentration-response curves after densitometric analyses of Western blots (Fig. 2D). ERK1/2 phosphorylation stimulated by norepinephrine was blocked by the MEK1/2 inhibitor PD 98059 (Fig. 3, A and B). MEK1/2 is a kinase that catalyzes ERK1/2 phosphorylation (Alessi et al., 1995). To show that norepinephrine-induced ERK1/2 phosphorylation in _1a-AR-transfected CHO-K1 cells is not due to stimulation of endogenously expressed -ARs, the ₁-AR antagonist prazosin, the -AR agonist isoproterenol, and the -AR antagonist propranolol were used to stimulate or block the effects of ₁- and -ARs. Norepinephrine-stimulated ERK1/2 phosphorylation in _1a-AR-transfected CHO-K1 cells was blocked by the ₁-AR antagonist prazosin but not by the -AR antagonist propranolol. The -AR agonist isoproterenol did not stimulate ERK1/2 phosphorylation (Fig. 3, C and D).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A, time course of norepinephrine (NE)-stimulated phosphorylation of ERK1/2 in 1a-AR-transfected CHO-K1 cells. The top lane of the immunoblots is the phosphorylated form of ERK1/2, whereas the bottom lane of immunoblots represents total ERK1/2 (phosphorylation state-independent). The 44 KD represents ERK1 and 42 KD represents ERK2. Cells were incubated with 10 µM NE for the indicated times and cellular lysates were immunoblotted with phospho-specific (top lane) and nonspecific (bottom lane) anti-ERK1/2 antibody, as described under Materials and Methods. A representative immunoblot from three independent experiments is shown. B, bands obtained in Western blots were analyzed by densitometry, and the optical densities ± S.E.M. from three experiments were plotted versus the incubation time with NE. C, concentration-dependent activation of ERK1/2 phosphorylation by NE in CHO-K1 cells expressing the mouse 1a-AR subtype. CHO-K1 cells were incubated with different concentrations of NE for 10 min and cellular lysates were immunoblotted with phospho-specific (top lane) and nonspecific (bottom land) anti-ERK 1/2 antibodies as described above. A representative immunoblot from four independent experiments is shown. D, bands obtained in Western blots were analyzed by densitometry, and the optical densities ± S.E.M. from four experiments were plotted as a percentage of the maximal NE response.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   A, effect of 50 µM PD 98059 (PD) on norepinephrine (NE)-induced phosphorylation of ERK1/2 in CHO-K1 cells expressing the mouse 1a-AR. Both phosphorylated (top lane) and total (bottom lane) immunoblots of anti-ERK1/2 from a representative experiment are shown. B, densities of bands from immunoblots were analyzed by densitometry and expressed as -fold stimulation over basal. The figure is the mean ± S.E.M. of three independent experiments. *, significantly different from other treatments (P < 0.05). C, NE-induced ERK1/2 phosphorylation in CHO-K1 cells transfected with 1a-ARs was not due to -ARs. The top lane of the immunoblots shows phosphorylated ERK1/2 from transfected CHO-K1 cells treated with 10 µM NE, 1 µM of the 1-AR antagonist prazosin (Pra), 1 µM of the -AR agonist isoproterenol (Iso), and 1 µM of the -AR antagonist propranolol (Prop), respectively. 1a-AR-transfected CHO-K1 cells were preincubated with antagonists for 1 h before they were treated with NE or isoproterenol for 10 min. The bottom lane of the immunoblots is total ERK1/2. The immunoblots are typical of three independent experiments. D, band densities from immunoblots were analyzed by densitometry and expressed as -fold stimulation over basal. The figure is the mean ± S.E.M. of three independent experiments. *, significantly different from other treatments (P < 0.05).

ERK1/2 Activation in _1a-AR-Transfected CHO-K1 Cells was Ca²⁺-Dependent and PKC-Independent. Because different investigators have reported opposite results regarding the Ca²⁺ dependence of ERK1/2 activation after _1a-AR stimulation in other cell types (Berts et al., 1999; Hu et al., 1999), we investigated whether norepinephrine-stimulated ERK1/2 phosphorylation required mobilization of Ca²⁺ in our _1a-AR-transfected CHO-K1 cells. Our strategy was to use the intracellular Ca²⁺ chelator BAPTA to prevent agonist-induced increases in intracellular Ca²⁺. To determine the conditions needed to chelate intracellular Ca²⁺ mobilized by norepinephrine, cells were preincubated with 10 or 50 µM BAPTA followed by norepinephrine stimulation. Preincubation with 10 µM BAPTA for 1 h partially inhibited the Ca²⁺ response to 10 µM norepinephrine, whereas preincubation with 50 µM BAPTA for 1 h abolished the Ca²⁺ response to norepinephrine (data not shown). Thus, we used 50 µM BAPTA in subsequent Western blotting experiments. Norepinephrine (10 µM) stimulated the phosphorylation of ERK1/2 and this effect was blocked by pretreating the cells with 50 µM BAPTA (Fig. 4A).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   A, norepinephrine (NE)-mediated phosphorylation of ERK1/2 in 1a-AR-transfected CHO-K1 cells was dependent on Ca2+ mobilization. Preincubation of 1a-AR-transfected CHO-K1 cells with the intracellular Ca2+ chelator BAPTA (50 µM, 1 h) blocked NE-stimulated phosphorylation of ERK1/2. Both phosphorylated (top lane) and total (bottom lane) immunoblots of anti-ERK1/2 from a representative experiment are shown. Data presented are typical of three experiments. B, NE-mediated phosphorylation of ERK1/2 in 1a-AR transfected CHO-K1 cells was independent of PKC. Cells were treated with 100 nM of the PKC inhibitor bisindolylmaleimide (Bis) for 1 h before stimulation with either 10 µM NE for 10 min or 100 nM PMA for 15 min. Note that PMA stimulation of ERK1/2 phosphorylation (receptor-independent phosphorylation) was blocked by preincubation with bisindolylmaleimide. Both phosphorylated (top lane) and total (bottom lane) immunoblots from a representative experiment are shown. Data presented are typical of three experiments.

Norepinephrine-Induced cAMP Accumulation in _1a-AR-Transfected CHO-K1 Cells. _1a-AR activation by 10 µM norepinephrine increased cAMP accumulation in _1a-AR-transfected CHO-K1 cells. This effect was blocked by the ₁-AR antagonist prazosin (Fig. 5A). To verify that native -AR receptors were not mediating this effect of norepinephrine, the -AR antagonist 1 µM propranolol was used, and as expected, did not alter norepinephrine-stimulated cAMP accumulation. In addition, after blocking ₁-ARs with prazosin, the -AR agonist isoproterenol did not stimulate cAMP accumulation in _1a-AR-transfected CHO-K1 cells (Fig. 5A), verifying the lack of functional -ARs. Prazosin and propranolol alone did not have any significant effect on cAMP accumulation (data not shown). Norepinephrine (10 µM) caused a rapid increase in cAMP accumulation in _1a-AR-transfected CHO-K1 cells with the maximum response at 2 min (Fig. 5B). Norepinephrine increased cAMP accumulation in a concentration-dependent manner (Fig. 5C) with an EC₅₀ value of 718.9 ± 15.97 nM. Because norepinephrine-stimulated ERK1/2 phosphorylation in _1a-AR-transfected CHO-K1 cells is dependent on a rise in intracellular Ca²⁺, we determined whether the increase in cAMP accumulation induced by norepinephrine was also dependent on intracellular Ca²⁺. We incubated _1a-AR-transfected CHO-K1 cells with the Ca²⁺ chelator BAPTA (50 µM) before stimulation with norepinephrine. Chelation of intracellular Ca²⁺ with BAPTA blocked _1a-AR-mediated cAMP accumulation (Fig. 5D). Chelation of intracellular Ca²⁺ with BAPTA also inhibited forskolin-stimulated (100 µM) cAMP accumulation by 69% (data not shown), suggesting that adenylyl cyclase activation is at least partially sensitive in these cells.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   A, cAMP accumulation in CHO-K1 cells transfected with 1a-ARs. Transfected CHO-K1 cells were pretreated with 1 µM prazosin (Pra) or 1 µM propranolol (Pro) for 1 h and then treated with 10 µM norepinephrine (NE) or 1 µM isoproterenol (Iso) for 10 min, or 100 µM forskolin (forsk) for 1 h as a positive control. cAMP was assayed by using a [3H]cAMP kit (Diagnostic Products). Data are the mean ± S.E.M. of three experiments. B, time course of NE-stimulated cAMP accumulation in 1a-AR-transfected CHO-K1 cells. Transfected CHO-K1 cells were treated with 10 µM NE for 0 and 30 s and for 1, 2, 5, 10, 20, and 30 min, and cAMP was assayed. Data are the mean ± S.E.M. of four experiments. C, concentration-response curve of NE-induced cAMP accumulation in transfected CHO-K1 cells. The cAMP responses induced by NE at each concentration were expressed as percentage of maximal response. Data are the mean ± S.E.M. of three experiments. D, NE-induced cAMP accumulation was blocked by the Ca2+ chelator BAPTA. Transfected CHO-K1 cells were treated with 50 µM BAPTA for 1 h then treated with 10 µM NE for 10 min. Data are the mean ± S.E.M. of three experiments. *, significantly different from other treatments (P < 0.05).

Effect of _1a-AR-Stimulated cAMP Accumulation on ERK1/2 Phosphorylation. Because norepinephrine stimulated both ERK1/2 phosphorylation and cAMP accumulation, we next investigated the interaction between these two pathways by studying the effects of adenylyl cyclase and protein kinase A activation on norepinephrine-induced ERK1/2 phosphorylation. Norepinephrine-stimulated ERK1/2 phosphorylation in _1a-AR-transfected CHO-K1 cells was enhanced by pretreating the cells with 50 and 100 µM of the adenylyl cyclase inhibitor SQ 22538 (Fig. 6, A and B) and 300 nM of the protein kinase A inhibitor CMQ (Fig. 6, C and D) for 1 h. Consistent with the results using inhibitors, ERK1/2 phosphorylation induced by norepinephrine was inhibited by 100 µM of the adenylyl cyclase activator forskolin. Forskolin alone did not have any significant effect on ERK1/2 phosphorylation (Fig. 6, C and D).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   A, norepinephrine (NE)-stimulated ERK1/2 phosphorylation was potentiated by the adenylyl cyclase inhibitor SQ 22536 (SQ) in CHO-K1 cells transfected with 1a-ARs. Transfected CHO-K1 cells were treated with 50 and 100 µM SQ 22536 for 1 h and then treated with 10 µM NE for 10 min. The top and bottom immunoblots are phosphorylated and total ERK1/2, respectively. These immunoblots are from a single experiment. B, densities of bands from immunoblots were analyzed by densitometry and expressed as -fold stimulation over basal. The figure is the mean ± S.E.M. of three independent experiments. *, significantly different from other treatments (P < 0.05). C, NE-stimulated ERK1/2 phosphorylation was enhanced by the protein kinase A inhibitor CMQ and inhibited by forskolin (forsk) in CHO-K1 cells transfected with 1a-ARs. Transfected CHO-K1 cells were treated with 300 nM CMQ and 100 µM forskolin for 1 h and then were treated with 10 µM NE for 10 min. The top and bottom immunoblots are phosphorylated and total ERK1/2, respectively. These immunoblots are from a single experiment. D, band densities from immunoblots were analyzed by densitometry and expressed as -fold stimulation over basal. The figure is the mean ± S.E.M. of three independent experiments. *, significantly different from NE treatment (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

₁-ARs have been shown to couple to several signal transduction pathways, including phosphoinositide hydrolysis, adenylyl cyclase (Exton, 1985; Schwinn et al., 1991), and MAPK activation (Michelotti et al., 2000). In this study, we transfected mouse _1a-AR cDNA into CHO-K1 cells and investigated _1a-AR-induced activation of the ERK1/2 phosphorylation pathway and cAMP accumulation, and the effect of cAMP on ERK1/2 phosphorylation. ERK1/2 was phosphorylated after stimulation of _1a-ARs and this effect was Ca²⁺-dependent and PKC-independent. _1a-AR activation also caused cAMP accumulation in a Ca²⁺-dependent manner. Norepinephrine-stimulated ERK1/2 phosphorylation was enhanced by the adenylyl cyclase inhibitor SQ 22536 and the PKA inhibitor CMQ. Although stimulation of _1a-ARs caused ERK1/2 activation, this response was tempered by simultaneous inhibition of ERK1/2 activation by cAMP.

To test the validity of our cell model, we performed a number of studies to verify that our transfected CHO-K1 cells expressed functional _1a-AR. Membranes isolated from transfected CHO-K1 cells revealed a pharmacological profile typical of the _1a-AR, given that the ₁-AR agonist prazosin and the _1a-AR-selective antagonist 5-methylurapidil displayed subnanomolar affinities, whereas the _1d-AR-selective antagonist BMY-7378 displayed a 100-fold lower affinity. These values are similar to those reported for transfected human and rat _1a-ARs (Ford et al., 1997) and to the mouse _1a-AR transiently transfected in COS cells (Xiao et al., 1998). Norepinephrine stimulation of _1a-ARs stimulated intracellular Ca²⁺ accumulation in a concentration-dependent manner, which was absent in untransfected CHO-K1 cells that lack endogenous ARs (Ford et al., 1997). These results suggested that _1a-ARs in _1a-AR-transfected CHO-K1 cells were functional and behaved similarly to native _1a-ARs. Thus, our stably transfected cell line is a good model to investigate the signal transduction pathways of mouse _1a-ARs.

_1a-ARs, like other G protein-coupled receptors, transduce signals through changes in intracellular concentrations of second messengers. It is well accepted that activation of ₁-ARs activates the guanine nucleotide regulatory proteins Gq/11, which induce the hydrolysis of phosphatidylinositol-4,5-bisphosphate by PLC into IP₃ and DAG (Graham et al., 1996). Both IP₃ and DAG play important roles as second messengers that increase intracellular Ca²⁺ concentration and activate various PKC isoforms, respectively. Recently, it has been shown that stimulation of _1a-ARs can activate MAPKs, although the precise mechanism is not clear (Michelotti et al., 2000). In our cell model, activation of _1a-ARs stimulated the phosphorylation of ERK1/2 in a time- and dose-dependent manner. Our time course data for ERK1/2 phosphorylation were similar to those reported for _1a-AR stimulation in human vascular smooth muscle cells, which endogenously express _1a-ARs (Hu et al., 1999). The EC₅₀ values for norepinephrine-induced ERK1/2 phosphorylation and Ca²⁺ mobilization were similar, suggesting that there was a concomitant increase in ERK1/2 phosphorylation and intracellular Ca²⁺ accumulation following _1a-ARs activation. Norepinephrine can also stimulate -ARs to activate ERK1/2 (Williams et al., 1998). CHO-K1 cells lack endogenous ARs (Ford et al., 1997), and we further confirmed that there are no functional -ARs in our _1a-AR-transfected CHO-K1 cells because the ₁-AR antagonist prazosin but not the -AR antagonist propranolol blocked norepinephrine-induced ERK1/2 phosphorylation. In addition, the -AR agonist isoproterenol did not cause ERK1/2 activation. Norepinephrine-stimulated ERK1/2 phosphorylation in _1a-AR-transfected CHO-K1 cells was blocked by the MEK inhibitor PD 98059 and the Ca²⁺ chelator BAPTA, suggesting that ERK1/2 phosphorylation was downstream of MEK and depended on increases in intracellular Ca²⁺. The reported dependence of MAPK activation upon PKC activation by ₁-ARs varies among cells (Romanelli and van de Werve, 1997; Berts et al., 1999; Hu et al., 1999; Snabaitis et al., 2000). In our studies PKC activation was not required for _1a-AR-stimulated phosphorylation of ERK1/2 in _1a-AR-transfected CHO-K1 cells. Similar to other studies, short-term treatment with the phorbol ester PMA caused an increase in ERK1/2 phosphorylation, indicating that PKC-mediated ERK1/2 activation can occur in CHO-K1 cells.

_1a-AR activation of ERK1/2 regulates a variety of functions in various cell types. For example, _1a-ARs stimulate hypertrophy in adult rat ventricular myocytes through the MEK1/2-ERK1/2 pathway (Xiao et al., 2001): _1a-AR activation increases mitogenesis in human smooth muscle cells through MAPK (Hu et al., 1999), ERK1/2 mediates _1a-AR-induced smooth muscle contraction (Dessy et al., 1998), and _1a-ARs induce differentiation in transfected PC12 cells (Williams et al., 1998). In transfected CHO-K1 cells, Keffel et al. (2000) showed that _1a-AR stimulation inhibited basal and growth factor-stimulated cell growth. We have shown that _1a-AR activation induced cell morphological changes through ERK1/2 in our transfected CHO-K1 cell model (Jiao et al., 2001).

₁-ARs have also been shown to increase intracellular cAMP formation (Johnson and Minneman, 1986; Atkinson and Minneman, 1991; Schwinn et al., 1991; Horie et al., 1995). Although it is well established that ₂-ARs are coupled to adenylyl cyclase through G_s, the precise pathway for ₁-AR-mediated cAMP accumulation is not clear. Some studies suggested that ₁-ARs can couple to G_s to elevate intracellular cAMP levels (Horie et al., 1995); other investigators have found that ₁-AR-regulated cAMP formation may not involve direct activation of adenylyl cyclase (Schwinn et al., 1991). Our data demonstrated that in _1a-AR-transfected CHO-K1 cells, norepinephrine-stimulated cAMP accumulation through ₁-ARs was dependent on a rise in intracellular Ca²⁺. Although there was no significant difference between the EC₅₀ values for norepinephrine-induced Ca²⁺ response and norepinephrine-induced ERK1/2 phosphorylation, the EC₅₀ value for norepinephrine-induced cAMP accumulation was significantly different from the other two, with a 6-fold lower potency. These results suggest that the pathway for norepinephrine-induced Ca²⁺ mobilization and ERK1/2 phosphorylation is distinct from the pathway for norepinephrine-induced cAMP accumulation. Thus, it is possible that norepinephrine also stimulates cAMP accumulation through G_s, in addition to G_q/11. This possibility is further supported by the fact that BAPTA inhibited forskolin-stimulated cAMP formation in our transfected _1a-AR CHO-K1 cells. This phenomenon was also observed in CHO cells transfected with the vasopressin receptor by others (Klingler et al., 1998). It has also been shown that -ARs can couple to G protein families other than G_q/11, such as in cardiac fibroblasts (Meszaros et al., 2000) and CHO-K1 cells (Horie et al., 1995).

We investigated the complex signal transduction pathways and interaction among these pathways in response to _1a-AR activation in transfected CHO-K1 cells. One interesting finding of this study is the inhibitory effect of cAMP on ERK1/2 phosphorylation after _1a-AR activation. Based on the data in this study, a model of the signal transduction pathways linked to _1a-AR activation in transfected CHO-K1 cells is proposed in Fig. 7. After _1a-AR activation, G_q/11 proteins are activated, leading to the stimulation of the phosphoinositide hydrolysis pathway that activates the MAPK module comprised of Raf, MEK, and ERK1/2. G_s may be activated as well, resulting in adenylyl cyclase activation. ERK1/2 phosphorylation is Ca²⁺-dependent and PKC-independent, whereas increases in cAMP are also dependent on the rise of intracellular Ca²⁺. The increase in cAMP activates PKA, which then inhibits the MAPK pathway. Our time course data for _1a-AR-stimulated cAMP accumulation, and our results using inhibitors of adenylyl cyclase and PKA show that this inhibitory effect on ERK1/2 activation occurs in parallel with the excitatory effect from PLC activation. Thus, the degree of ERK1/2 phosphorylation after _1a-AR stimulation is the net product of activation by PLC and inhibition by adenylyl cyclase. At present it is uncertain whether the dual regulation of ERK1/2 described herein is functionally significant in cells and tissues in vivo. It is possible that either the overexpression of the _1a-ARs and/or the CHO-K1 cell phenotype have provided a milieu that enables observation of this interaction. However, because _1a-AR-mediated PLC, ERK1/2 and adenylyl cyclase stimulation has been observed in native tissues, it seems likely that cross talk between these pathways could occur in vivo. The recent discovery of multiple isoforms of the _1a-AR (Coge et al., 1999) also raises the possibility that these distinct isoforms could interact differently with other signaling molecules (e.g., G_s and G_q/11), creating an array of possible combinations for regulation of function in various cells and tissues. Further investigation is needed to understand the mechanisms and physiological significance of the interaction between these signaling pathways.

View larger version (8K): [in this window] [in a new window]   Fig. 7.   Proposed scheme of norepinephrine induced 1a-AR activation in transfected CHO-K1 cells. Stimulation of 1a-ARs with norepinephrine (NE) activates the G protein Gq/11, which in turn activates the phospholipase hydrolysis pathway, which then increases intracellular Ca2+ and activates PKC. Stimulation of 1a-ARs may also cause activation of the G protein Gs, which stimulates adenylyl cyclase. The Raf-MEK-ERK1/2 module and the adenylyl cyclase pathway are activated by increase in intracellular Ca2+. cAMP activates PKA, which inhibits the activation of ERK1/2. The cellular effects of 1a-AR stimulation of the Raf-MEK-ERK1/2 module are modulated by an interaction between the PLC signaling pathway and the AC signaling pathway.