Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment

Charles W. Emala1, Judith Clancy-Keen2, and Carol A. Hirshman1

1 Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 2 Department of Environmental Health Sciences, Division of Physiology, Johns Hopkins School of Public Health, Baltimore, Maryland 21205


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either Gsalpha or adenylyl cyclase. Detergent-solubilized Gsalpha from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) when reconstituted into S49 cyc- membranes that lack endogenous Gsalpha (carbachol pretreated: GTPgamma S, 93 ± 13% of control; NaF/AlCl3, 99 ± 8.6% of control; n = 4). Exogenous Gsalpha solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 ± 10% of control; NaF/AlCl3, 54 ± 11% of control; n = 4). [3H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.

muscarinic; [3H]forskolin; reconstitution; S49 cyc-; bovine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY SMOOTH MUSCLE FROM asthmatic patients (4) and from animal models of asthma (11) show reduced relaxation in response to beta -adrenoceptor agonists. The distal effector targets of beta -adrenoceptor stimulation are the adenylyl cyclase enzymes. This enzyme family consists of at least nine subtypes that differ in their responses to products of other second messenger pathways, including calcium (6, 24, 28, 31), G protein beta gamma -subunits (5, 27, 33), protein kinase A (13), and protein kinase C (PKC; see Refs. 14, 15, 17). Evidence to date suggests that reduced beta -adrenoceptor responsiveness in asthmatic airway smooth muscle is not only due to changes at the level of the beta -adrenoceptor but often involves more distal components of this receptor-Gsalpha -adenylyl cyclase signaling cascade (3, 10).

The airway inflammatory component of asthma results in elevated levels of agonists such as histamine and bradykinin and increased release of acetylcholine, all of which activate airway smooth muscle receptors that couple to the Gq/PKC signaling pathway. It is known that PKC can acutely regulate the function of some adenylyl cyclase subtypes (14, 15, 17). Thus chronic activation of the Gq/PKC pathway is one possible mechanism by which airway smooth muscle relaxation in response to agonists that increase cAMP may be impaired in asthma.

We have previously shown that chronic but not acute activation of muscarinic receptors results in downregulation of adenylyl cyclase activity in response to GTP or forskolin, an effect blocked by the protein kinase inhibitor staurosporine, but the site (i.e., Gsalpha or adenylyl cyclase) of this effect was never fully identified (23). Neither decreased numbers of cell surface beta -adrenoceptors nor increased quantity of Gialpha proteins could account for carbachol's effect since they were not changed. Because staurosporine blocked the effect of chronic carbachol activation, we reasoned that chronic activation of the M3 muscarinic/Gq/PKC pathway might directly downregulate the function and/or expression of the adenylyl cyclase protein. Direct downregulation of adenylyl cyclase protein as opposed to decreased activity has never been demonstrated in response to chronic muscarinic receptor activation in any cell.

In our previous study, chronic muscarinic receptor activation reduced subsequently measured forskolin-stimulated adenylyl cyclase activity via a staurosporine-sensitive pathway. Although it is generally believed that forskolin directly stimulates adenylyl cyclase activity, the resulting level of activation is dependent upon the activation state of Gsalpha (26). Thus forskolin is not a selective measure of adenylyl cyclase function only. Before more directed mechanistic studies are planned to evaluate the potential for direct regulation of the expression of specific subtypes of adenylyl cyclases by chronic muscarinic receptor activation in airway smooth muscle, one must unequivocally implicate adenylyl cyclase as the target of muscarinic receptor-induced downregulation.

The present study was therefore designed to determine whether chronic activation of muscarinic receptors regulates protein expression or function at the level of either the Gsalpha protein or the adenylyl cyclase enzymes. We used complementary Gsalpha reconstitution studies to show that Gsalpha extracted from chronic muscarinic receptor-activated airway smooth muscle had similar function when reconstituted into S49 cyc- membranes and that reconstitution of exogenous Gsalpha back into airway smooth muscle failed to restore normal adenylyl cyclase activity after chronic muscarinic receptor activation. Finally, we were able to directly measure the total amount of adenylyl cyclase protein by radioligand binding and show for the first time in any cell that chronic muscarinic receptor activation leads to downregulation in the function and expression of adenylyl cyclase protein.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airway smooth muscle preparation and pretreatment protocol. Bovine airway smooth muscle was isolated from tracheas obtained from a local abattoir by previously described methods (23). Briefly, airway smooth muscle was removed from the trachea and finely minced with razor blades and then was washed two times in serum-free DMEM. These small airway smooth muscle fragments (~1 × 1 mm) were incubated for the indicated period of time with the indicated pretreatment in serum-free DMEM plus antibiotics (100 U/ml penicillin G, 100 µg/ml streptomycin, 0.025 µg/ml amphotericin B, 100 U/ml nystatin, and 1 µg/ml gentamicin) at 37°C in an atmosphere of 5% CO2-95% air. Untreated control tissues were incubated for the same length of time in parallel experiments to compensate for any loss of adenylyl cyclase activity due to incubation time. Airway smooth muscle fragments were recovered at the same time from control and pretreated groups after incubations and were washed three times in serum-free cell culture media to remove excess effectors used for pretreatments. Muscle was then homogenized using high-speed cutting blades (Tissumizer Mark II; Tekmar-Dohrmann, Cincinnati, OH) at top speed for 30 s on ice, filtered through one layer of Nitex mesh, and frozen at -70°C for subsequent use in adenylyl cyclase, [3H]forskolin radioligand binding, and protein assays. Protein assays were performed immediately before adenylyl cyclase or radioligand binding assays, and control and treated tissues were always analyzed in parallel within the same binding or adenylyl cyclase assay.

Adenylyl cyclase assays in native bovine airway smooth muscle membranes. Adenylyl cyclase activity was determined in triplicate samples as previously described (11), with minor modifications. Briefly, cellular lysates (40 µg) prepared from airway smooth muscle fragments that had been incubated for 18 h in the presence or absence of 10 µM carbachol were incubated for 10 min at 30°C in 100 µl final volume containing 50 mM HEPES (pH 8.0), 50 mM NaCl, 0.4 mM EGTA, 1 mg/ml BSA, 0.5 mM MgCl2, 0.1 mM [alpha -32P]ATP (0.1-0.3 mCi/µmol), 1 mM cAMP, 1 mM dithiothreitol (DTT), 7 mM creatine phosphate, and 5 units creatine phosphokinase. Other modulators were added as indicated in the text. Assay tubes containing isoproterenol and/or PGE1 also contained 10 µM GTP. Assay tubes containing 10 mM NaF also contained 100 µM AlCl3. Preliminary experiments confirmed the linearity of adenylyl cyclase activity at the protein concentrations and incubation times. cAMP was isolated by sequential chromatography through Dowex and alumina (22) with column recoveries of 75-90%. Basal and stimulated adenylyl cyclase activities are expressed as picomoles of cAMP synthesized per milligram membrane protein for the 10-min incubation period. Preliminary experiments were performed in the presence or absence of 100 µM atropine to confirm that adenylyl cyclase activity was not affected by residual carbachol used during pretreatment periods.

Preparation of cyc- membranes. Murine S49 lymphoma cyc- kinase+ cells were grown in spinner flasks in DMEM with high glucose, 10% horse serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Three liters of cells at a density of 5 × 106 cells/ml were used for membrane preparation. Cells were pelleted by centrifugation at 400 g for 15 min at 4°C and were resuspended in cold 0.1 M HEPES, pH 7.4 (40 ml buffer/l of cells). Cells were disrupted by 30 strokes of a motor-driven Teflon pestle in a glass homogenizer. Undisrupted cells and large organelles were removed by centrifugation at 400 g for 15 min at 4°C, and the supernatant containing cyc- cell membranes was stored in 1-ml aliquots at -70°C until used for reconstitution assays.

Preparation of red blood cell membranes. Outdated human red blood cells (RBCs) were obtained from a local blood bank and stored at 4°C. Fifty milliliters of packed RBCs were mixed with 150 ml cold PBS (0.144 g/l KH2PO4, 9.0 g/l NaCl, and 0.795 g/l Na2HPO4 · 7H2O), pH 7.4, and centrifuged at 5,900 g for 10 min at 4°C. The supernatant was discarded, and cells were washed with 150 ml cold PBS, centrifuged as above, and resuspended in 50 ml of 5 mM Na2HPO4, pH 8.0, for hypotonic cell lysis. Lysed cells were centrifuged at 25,000 g for 15 min at 4°C, after which the supernatant was gently aspirated and discarded. The RBC membrane pellet was repeatedly washed (4-5 times) with 5 mM Na2HPO4 until the pellet appeared white (indicating removal of Hb), and membranes were frozen in 1-ml aliquots at -70°C at 3-4 mg/ml.

Detergent extraction of Gsalpha from human RBCs or bovine airway smooth muscle. Gsalpha was detergent solubilized from human RBCs or bovine airway smooth muscle membranes by modifications of previously published methods (7, 8). The day before the adenylyl cyclase reconstitution assay, RBC or bovine airway smooth muscle membranes were thawed and microcentrifuged at 14,000 g for 30 min at 4°C. The pellet was resuspended at 3 mg/ml in 1 ml of TEM-Lubrol-DTT (final concentrations; 10 mM Tris, pH 7.5, 0.1 mM EDTA, 10 mM MgCl2, 0.2% Lubrol PX, and 1 mM DTT) and incubated overnight at 4°C with gentle rocking. Microcentrifuge tubes were centrifuged in an SS34 rotor (Sorvall) at 20,000 rpm [minimum radius (rmin) g force of 14,300 g] for 30 min at 4°C, and the supernatant containing solubilized RBC or bovine airway smooth muscle Gsalpha was saved and divided into two aliquots. One aliquot containing functional Gsalpha was kept on ice and labeled the soluble extract. The second aliquot was heated to 65°C for 60 min to inactive Gsalpha and was labeled heated extract (negative control). Additional 50-µl aliquots from control and carbachol-pretreated samples were subjected to immunoblot analysis to determine the content of Gsalpha .

Reconstitution of RBC Gsalpha into bovine airway smooth muscle membranes and measurement of adenylyl cyclase activity. Assay tubes were assembled in triplicate in a final volume of 80 µl. This reaction consisted of 20 µl of bovine airway smooth muscle membranes (2 mg/ml), 10 µl of either soluble extract (containing functional Gsalpha ) or heated extract (containing inactive Gsalpha ), and 50 µl of cold assay buffer (final concentrations 50 mM Tris, pH 7.6, 1 mM EDTA, 1 mM DTT, 2 mM MgCl2, 0.1 mM ATP, 0.2 mM cAMP, 6.7 mM creatine phosphate, and 50 U/ml creatine phosphokinase) containing the indicated effectors [achieving a final concentration of either 10 mM NaF/100 µM AlCl3, 10 µM GTP, or 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S)]. Reconstitution of solubilized RBC Gsalpha into control or carbachol-pretreated bovine airway smooth muscle membranes was allowed to occur at 37°C for 1 h, after which time 20 µl of [alpha -32P]ATP (20-30 µCi/ml, 800 Ci/mmol) were added to each tube, and the incubation continued for an additional 20 min. The reaction was stopped by the addition of 100 µl stop buffer [50 mM HEPES, pH 7.5, 2 mM ATP, 0.5 mM cAMP, 2% SDS, and 1 µCi/ml [3H]cAMP (37 Ci/mmol)], and newly synthesized [32P]cAMP was separated from [alpha -32P]ATP using [3H]cAMP to monitor column recovery by sequential column chromatography over Dowex and alumina according to the method of Salomon et al. (22). Preliminary experiments established that the time and temperature of incubation resulted in linear increases in adenylyl cyclase activity and that 10 µl/tube of soluble extract resulted in maximal adenylyl cyclase activity for a given effector in both control and carbachol-pretreated bovine airway smooth muscle membranes. Adenylyl cyclase activity was expressed as picomole cAMP per milligram membrane protein per 20 min, and activity in carbachol-pretreated membranes was expressed as percentage of activity in control membranes. Positive control experiments were performed in parallel using soluble and heat-inactivated extracts of RBCs reconstituted into cyc- membranes to ensure successful reconstitution within each experiment.

Reconstitution of bovine airway smooth muscle Gsalpha into cyc- membranes and measurement of adenylyl cyclase activity. Assay tubes were assembled in triplicate in a final volume of 80 µl. Each tube contained 10 µl of either soluble or heated-inactivated bovine airway smooth muscle detergent extract, 20 µg of cyc- membranes, and 50 µl of cold assay buffer with effectors (see above). Reconstitution of solubilized bovine airway smooth muscle Gsalpha into cyc- membranes was allowed to occur at 37°C for 1 h, after which 20 µl of [alpha -32P]ATP (20-30 µCi/ml, 800 Ci/mmol) were added to each tube and the incubation was continued for an additional 20 min. The reaction was terminated, and [32P]cAMP was isolated exactly as described above. Preliminary experiments established that the time and temperature of incubation resulted in linear increases in adenylyl cyclase activity. Adenylyl cyclase activity was expressed as picomole cAMP per milligram membrane protein per 20 min, and adenylyl cyclase activity in cyc- membranes reconstituted with soluble extracts from carbachol-pretreated bovine airway smooth muscle membranes was expressed as percentage of activity in cyc- membranes reconstituted with soluble extracts from control membranes. Negative control experiments measured adenylyl cyclase activity in cyc- membranes in the absence of reconstitution to measure background adenylyl cyclase activity.

[3H]forskolin saturation radioligand binding. Bovine airway smooth muscle lysates (250 µg) from either control or carbachol-pretreated samples were incubated with [3H]forskolin (30.5 Ci/mmol; 4-120 nM) in duplicate in the presence or absence of 20 µM unlabeled forskolin in binding buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Cytochalasin B (40 µM) was also included in binding buffer to prevent nonspecific binding of [3H]forskolin to the glucose transporter (20). Gpp(NH)p (100 µM) and NaF (10 mM) were also included in binding buffer to maximize affinity of adenylyl cyclase for forskolin (2, 18). All radioligand binding experiments were incubated in a final volume of 125 µl and were performed for 2 h at room temperature. Preliminary experiments confirmed that equilibrium binding had been achieved using this time and temperature of incubation and that linear increases in specific binding occurred over the range of protein concentrations used. Preliminary studies also confirmed that saturation of [3H]forskolin specific binding occurred at 40 nM [3H]forskolin. Binding assays were rapidly terminated by the addition of 5 ml of cold wash buffer (50 mM Tris, pH 7.5, 10 mM MgCl2) followed by immediate vacuum filtration through GF/C glass fiber filters. Tubes were rapidly washed an additional three times with 5 ml cold wash buffer. Incorporation of 3H was determined by submerging filters in 5 ml of scintillation cocktail and counting in a Beckman scintillation counter with an efficiency of 50-55%. Specific binding (total binding in the absence of unlabeled forskolin minus binding in the presence of unlabeled forskolin) was calculated after subtraction of control counts obtained from tubes lacking membranes. Specific binding was plotted as a function of radioligand and was fitted to a rectangular hyperbola equation. Total receptor numbers (Bmax) were determined from the y plateau values, and affinity (Kd) was determined from the midpoint on the y-axis relative to the y plateau values. Binding data were then subjected to Scatchard-Rosenthal transformation of the data.

Immunoblot analysis of Gsalpha in soluble extracts of bovine airway smooth muscle. Fifty-microliter aliquots of Lubrol extracts of bovine airway smooth muscle were subjected to immunoblot analysis as previously described (12). Briefly, aliquots were mixed with an equal volume of gel loading buffer and after boiling for 10 min were subjected to electrophoresis through 10% polyacrylamide [10% total (T):0.1% crosslinker (C)]-SDS gels and transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked, PVDF membranes were incubated for 2 h in Gsalpha primary antibody (RM/1, 1:1,000 dilution; NEN, Boston, MA). After two 15-min washes, PVDF membranes were incubated for 2 h with goat anti-rabbit IgG-horseradish peroxidase (1:3,000 dilution; Amersham, Arlington Heights, IL). Bands were detected by enhanced chemiluminescence according to the manufacturer's recommendations (Immunolite II; Bio-Rad, Hercules, CA) with subsequent exposure to autoradiography film. Band intensities were quantified with a scanner coupled to a personal computer with MacBas 2.2 software.

Protein determination. Protein was assayed with the Pierce Chemical (Rockford, IL) BCA protein assay reagent, consisting of bicinchoninic acid and copper sulfate solutions (25). BSA was used as a standard.

Statistics. Unless otherwise noted, data are expressed as means ± SE. Data from adenylyl cyclase assays were analyzed by two-way ANOVA for treatments in the same tissues and one-way ANOVA for treatments between groups with Tukey-Kramer multiple comparisons. Data from forskolin binding assays were analyzed by two-tailed paired t-test. The null hypothesis was rejected when a P value < 0.05 was obtained.

Reagents. Except as noted, all reagents were purchased from Sigma (St. Louis, MO). All radioisotopes were purchased from NEN.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Adenylyl cyclase activity was compared in homogenates of bovine airway smooth muscle fragments after incubation for 18 h in the presence or absence of 10 µM carbachol. As we have previously shown (23), carbachol pretreatment resulted in significant decreases in basal and GTP-, isoproterenol-, and forskolin-stimulated adenylyl cyclase activity (Fig. 1). Control basal activities of 37 ± 9.9 pmol cAMP · mg protein-1 · 10 min-1 were reduced by carbachol pretreatment to 16 ± 5.7 pmol cAMP · mg protein-1 · 10 min-1 (57% decrease). Control GTP-stimulated activities of 51 ± 15 pmol cAMP · mg protein-1 · 10 min-1 were reduced by carbachol pretreatment to 20 ± 5.5 pmol cAMP · mg protein-1 · 10 min-1 (56% decrease). Control isoproterenol-stimulated activities of 90 ± 17 pmol cAMP · mg protein-1 · 10 min-1 were reduced by carbachol pretreatment to 33 ± 7.8 pmol cAMP · mg protein-1 · 10 min-1 (63% decrease). Control forskolin-stimulated activities of 121 ± 23 pmol cAMP · mg protein-1 · 10 min-1 were reduced by carbachol-pretreatment to 41 ± 15 pmol cAMP · mg protein-1 · 10 min-1 (66% decrease; n = 4).


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Fig. 1.   Adenylyl cyclase activity in bovine airway smooth muscle homogenates after 18 h of 10 µM carbachol pretreatment. Adenylyl cyclase activity under basal and GTP-, isoproterenol (Iso)-, or forskolin-stimulated conditions was decreased compared with control tissues stimulated with the same effectors (n = 4 experiments). * P < 0.05.

To confirm that the carbachol-induced decreases in adenylyl cyclase activity were not due to nonspecific cell toxicity resulting in an overall decline in enzyme activity, a separate group of experiments was performed in which carbachol was withdrawn after 18 h of incubation and 100 µM atropine was added for an additional 48 h. Recovery of adenylyl cyclase activities to control levels would suggest that the cells were not nonspecifically injured by the 18-h carbachol treatment. In these experiments (n = 3), homogenates prepared from airway smooth muscle fragments that had received carbachol for 18 h followed by 48 h of atropine had similar adenylyl cyclase activity to those cells maintained in parallel incubations without treatment for 66 h. Tissues treated for 18 h with carbachol followed by 48 h of atropine had basal activities 88 ± 5% of control, GTP-stimulated activities were 90 ± 18% of control, isoproterenol-stimulated activities were 104 ± 11% of control, and forskolin-stimulated activities were 125 ± 26% of control (Fig. 2). Incubation in the presence of atropine alone had no significant effect on adenylyl cyclase activity (data not shown).


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Fig. 2.   Recovery of adenylyl cyclase activity in bovine airway smooth muscle homogenates after an initial decrease after 18 h of 10 µM carbachol pretreatment. Carbachol-pretreated bovine airway smooth muscle showed significant decreases in adenylyl cyclase activity under basal and GTP-, isoproterenol-, and forskolin-stimulated conditions, which was restored to control values by washing away carbachol and incubating tissues for an additional 48 h in the presence of atropine (n = 3). * P < 0.05 compared with control tissues incubated in parallel without carbachol.

To determine where carbachol was having its effect in decreasing the activity of the receptor-G protein-adenylyl cyclase cascade, we performed a series of experiments to evaluate the quantity and function of the Gsalpha and adenylyl cyclase proteins. The function of Gsalpha was evaluated by complimentary reconstitution assays. In the first approach, Gsalpha was detergent solubilized from control or carbachol-pretreated bovine airway smooth muscle and reconstituted into a membrane system lacking endogenous Gsalpha (i.e., cyc-), and its function was assessed by stimulation with GTPgamma S and NaF/AlCl3. In a second complementary approach, Gsalpha was solubilized from human RBCs and reconstituted into control or carbachol-pretreated bovine airway smooth muscle to test for decreased Gsalpha function after carbachol pretreatment.

Activity levels of Gsalpha from control and carbachol-pretreated bovine airway smooth muscle reconstituted into cyc- cell membranes were not different. Adenylyl cyclase activity stimulated with 10 µM GTPgamma S was 93 ± 13% of control, whereas activity stimulated with 10 µM NaF/AlCl3 was 99 ± 8.6% of control (n = 4; Fig. 3A). Control experiments confirmed successful reconstitution of functional Gsalpha activity into cyc- membranes. The cyc- membranes had barely detectable adenylyl cyclase activity in the presence of NaF/AlCl3 or GTPgamma s (2.4 ± 1.4 and 1.1 ± 0.9 pmol cAMP · mg protein-1 · 10 min-1, respectively). After reconstitution with Gsalpha extracted from control bovine airway smooth muscle, adenylyl cyclase activity in response to NaF/AlCl3 and GTPgamma s increased to 222 ± 51 and 372 ± 68 pmol cAMP · mg protein-1 · 10 min-1, respectively (Fig. 3B). The quantity of Gsalpha solubilized from control and carbachol-pretreated bovine airway smooth muscle was not different, as detected by immunoblot analysis (Fig. 4).


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Fig. 3.   Adenylyl cyclase activity in cyc- membranes (memb) reconstituted with Gsalpha extracted from bovine airway smooth muscle. A: bovine airway smooth muscle was untreated (control) or treated for 18 h with 10 µM carbachol. After homogenization of smooth muscle, Gsalpha was detergent extracted and reconstituted into cyc- membranes lacking endogenous Gsalpha (see METHODS). Adenylyl cyclase activity was measured in response to activators of Gsalpha , guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), or NaF/AlCl3. Reconstituted Gsalpha from control or carbachol-pretreated bovine airway smooth muscle had similar activities when reconstituted into cyc- membranes (n = 4). B: control experiments were performed to ensure successful reconstitution of bovine Gsalpha using cyc- membranes. Adenylyl cyclase activity in cyc- membranes increased in response to 10 mM NaF/AlCl3 after reconstitution (n = 4). * P = 0.01.



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Fig. 4.   Representative immunoblot detection of Gsalpha in solubilized extracts of control and carbachol-pretreated bovine airway smooth muscle. Bovine airway smooth muscle was untreated (control) or treated for 18 h with 10 µM carbachol. After homogenization of smooth muscle, Gsalpha was detergent extracted, and equal aliquots were subjected to immunoblot analysis. The quantity of Gsalpha was not different in extracts from control vs. carbachol pretreatment. Shown is a representative experiment of 6.

Gsalpha was solubilized from human RBCs and reconstituted into control or carbachol-pretreated bovine airway smooth muscle homogenates in an attempt to restore normal adenylyl cyclase activity to carbachol-pretreated muscle. However, decreases in basal and GTP- and NaF/AlCl3-stimulated adenylyl cyclase activity persisted in carbachol-pretreated bovine airway smooth muscle after reconstitution. GTP- and NaF/AlCl3-stimulated activities were 36 ± 10 and 54 ± 11% of control activities, respectively (n = 4; Fig. 5A). Control experiments were performed in parallel in cyc- membranes to ensure successful reconstitution of RBC Gsalpha . NaF/AlCl3-stimulated adenylyl cyclase activity was 9.3 ± 2.1 and 193 ± 42 in cyc- membranes in the absence and presence of solubilized RBC Gsalpha , respectively (n = 4; Fig. 5B).


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Fig. 5.   Adenylyl cyclase activity in bovine airway smooth muscle homogenates after reconstitution with Gsalpha extracted from red blood cells (RBC). A: bovine airway smooth muscle was untreated (control) or treated for 18 h with 10 µM carbachol. After homogenization of smooth muscle, exogenous Gsalpha extracted from RBCs was reconstituted in bovine airway smooth muscle homogenates, and adenylyl cyclase activity was measured in response to activators of Gsalpha . Decreased adenylyl cyclase activity in response to 10 µM GTP or 10 mM NaF persisted in carbachol-pretreated bovine airway smooth muscle compared with control despite reconstitution of functional Gsalpha from RBCs (n = 4). * P < 0.01. B: control experiments were performed to ensure successful reconstitution of RBC Gsalpha using cyc- membranes. Adenylyl cyclase activity in cyc- membranes increased in response to 10 mM NaF after reconstitution (n = 4) * P = 0.01.

Radioligand binding assays were performed to measure the quantity of total adenylyl cyclase enzyme in control and carbachol-pretreated bovine airway smooth muscle. A significant reduction in adenylyl cyclase protein was measured in carbachol-pretreated bovine airway smooth muscle compared with control (Bmax = 107 ± 31 and 152 ± 40 fmol/mg protein, respectively; n = 5; P = 0.02; Fig. 6). Saturation of specific binding was achieved at 40 nM of [3H]forskolin (Fig. 7A), and specific binding (Bmax) and affinity values (Kd) were determined from nontransformed data before Scatchard-Rosenthal transformations of the data were performed, which revealed a single-affinity binding site (Fig. 7B). The affinity (Kd) of [3H]forskolin for its binding site was not different in control and carbachol-pretreated bovine airway smooth muscle, with Kd values of 52 ± 8.0 and 48 ± 12 nM, respectively.


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Fig. 6.   Total specific binding (Bmax) in control or carbachol-pretreated bovine airway smooth muscle homogenates. Carbachol pretreatment (10 µM for 18 h) resulted in a significant decrease in the expression of total adenylyl cyclase proteins (n = 5). P = 0.02.



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Fig. 7.   Representative saturation radioligand binding isotherm of [3H]forskolin in bovine airway smooth muscle homogenates. A: saturation of specific binding was achieved over the range of [3H]forskolin used. B: representative Scatchard-Rosenthal transformation of saturation binding indicating a single class of binding sites.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the current study, bovine airway smooth muscle pretreated with carbachol for 18 h showed normal Gsalpha function in reconstitution assays but reduced adenylyl cyclase activity and reduced amounts of total adenylyl cyclase protein. This study extends previous studies from our laboratory demonstrating that chronic carbachol pretreatment resulted in decreased adenylyl cyclase activity by implying that the site of regulation was at the level of adenylyl cyclase protein expression and not Gsalpha .

In the current study, reconstitution of Gsalpha from an exogenous source (human RBC) into carbachol-pretreated bovine airway smooth muscle failed to restore normal adenylyl cyclase activities. Additionally, Gsalpha extracted from carbachol-pretreated bovine airway smooth muscle and reconstituted into a membrane preparation lacking endogenous Gsalpha (i.e., S49 cyc- cells) had normal activity in this alternate membrane environment compared with Gsalpha extracted from control bovine airway smooth muscle. The results taken together suggest that carbachol pretreatment was having an effect at the level of the adenylyl cyclase enzyme itself. Therefore, radioligand binding assays were performed to determine if quantitative changes occurred in adenylyl cyclase protein. These studies confirmed a decrease in the amount of total adenylyl cyclase protein after carbachol pretreatment of bovine airway smooth muscle that likely accounts for the decrease in adenylyl cyclase activity in response to effectors acting at the receptor and Gsalpha protein levels of this signaling cascade.

Carbachol-induced decreases in adenylyl cyclase activity could not be due to loss of adenylyl cyclase enzyme activity during the 18-h incubation period, since control tissues incubated in parallel under identical conditions exhibited increased levels of adenylyl cyclase compared with carbachol-pretreated tissues. Carbachol-induced decreases in adenylyl cyclase activity were not due to nonspecific cellular toxicity induced by carbachol, since withdrawal of the carbachol and further incubation for 48 h in the presence of atropine allowed recovery of adenylyl cyclase activity to control levels.

Previous studies in a variety of cell lines agree with the current study in that chronic activation of either PKC with phorbol esters or of receptors potentially coupled to Gqalpha leads to decreased function of the receptor-Gsalpha -adenylyl cyclase cascade. However, this is the first study in any cell to show a reduced amount of adenylyl cyclase protein after chronic muscarinic receptor activation. In the osteoblast-like cell line UMR-106, >6 h of pretreatment with phorbol esters resulted in decreased adenylyl cyclase activity in the presence of parathyroid hormone or forskolin, suggesting PKC-mediated downregulation of adenylyl cyclase function (16). In contrast, a study in canine cultured thyroid cells showed that 18 h of phorbol ester pretreatment resulted in decreased adenylyl cyclase activity in response to thyroid-stimulating hormone but not forskolin (9), suggesting PKC cross talk at a stimulatory receptor/Gsalpha level. However, in a subsequent study, these authors showed that 2-4 h of carbachol pretreatment (presumably working through a muscarinic receptor-Gqalpha -PKC pathway in these cells) resulted in decreased adenylyl cyclase activity in response to thyroid-stimulating hormone, cholera toxin, GTP, and forskolin, again suggesting cross-regulation at the level of adenylyl cyclase (21). In none of these studies were Gsalpha reconstitution studies or measurement of adenylyl cyclase protein performed. Perhaps these studies differed regarding the apparent site of PKC cross-regulation because 1) phorbol ester may stimulate more than PKC (shown to also stimulate mitogen-activated protein kinases; see Refs. 1 and 30), 2) the phorbol ester pretreatment was for a different time course than carbachol pretreatment, 3) different isoforms of PKC may exist in the different cell types or be activated/downregulated differently by phorbol esters, or 4) activation of Gqalpha by receptor occupancy may couple to other signaling pathways besides PKC.

The current study also agrees with a recently published study from our laboratory using myometrial cells demonstrating that chronic treatment of cultured rat myometrial cells with either oxytocin or phenylephrine (both coupled to Gqalpha ) resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, NaF, MnCl2, or forskolin (suggesting cross-regulation at the level of adenylyl cyclase) by a mechanism that was blocked by prior depletion of PKC by prolonged phorbol ester treatment (19). Thus these studies in a variety of cell lines coupled with the results of the present study suggest that Gqalpha /PKC activation potentially cross-regulates several levels of the receptor-Gsalpha -adenylyl cyclase cascade, but the present study is the first study to demonstrate a downregulation of adenylyl cyclase protein.

Classically, dual regulation of adenylyl cyclase activity has been described when Gsalpha - or Gialpha -coupled receptors are acutely activated. However, chronic activation of Gi-coupled receptors is known to upregulate subsequently measured adenylyl cyclase activity in many cell types and is known as adenylyl cyclase superactivation. In studies evaluating the phenomenon of adenylyl cyclase "superactivation," it is apparent that adenylyl cyclase enzymes differ in both their acute and their chronic response to Gi-coupled receptor activity. In COS-7 cells transfected with various subtypes of adenylyl cyclase, patterns of responses to acute or chronic Gi activation were seen that coincided with previous subfamily groupings of the adenylyl cyclase family (29). Subtypes I, V, VI, and VIII were acutely inhibited by dopamine D2 receptor activation (coupling through Gi), but chronic dopamine D2 receptor activation resulted in superactivation of these enzymes' activities. Subtypes II, IV, and VII were activated by acute D2 receptor activation but were inhibited after prolonged D2 receptor occupancy. Conversely, type III was inhibited by either acute or chronic D2 receptor activation. Although these findings are not directly linked to the current study in which muscarinic receptor activation is involved, it suggests that different subtypes of adenylyl cyclases differ in their responses to chronic Gq/PKC activation, which may account for different effects of chronic Gq/PKC stimulation found in different cell types. Indeed, isoforms II, III, V, and VII are activated by acute treatments with phorbol esters (14, 15, 32), and subtypes II and V are known to be phosphorylated by PKC-alpha (15, 34). Perhaps a desensitization mechanism exists for the downregulation of adenylyl cyclase subtypes sensitive to PKC-mediated phosphorylation under chronic PKC activation conditions.

In summary, chronic activation of muscarinic receptors by carbachol in airway smooth muscle downregulated the expression and function of adenylyl cyclase proteins with no functional effect on Gsalpha . Reduced adenylyl cyclase content of airway smooth muscle in response to chronic elevations of inflammatory agonists that couple to Gq in asthmatic airways may contribute to impaired relaxation of asthmatic airway smooth muscle in response to beta -agonists.


    FOOTNOTES

Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St. PH 525, New York, NY 10032 (E-mail: cwe5{at}columbia.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.

Received 5 November 1999; accepted in final form 17 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aharonovitz, O, and Granot Y. Stimulation of mitogen-activated protein kinase and Na+/H+ exchanger in human platelets. Differential effect of phorbol ester and vasopression. J Biol Chem 271: 16494-16499, 1996[Abstract/Free Full Text].

2.   Alousi, AA, Jasper JR, Insel PA, and Motulsky HJ. Stoichiometry of receptor-Gs-adenylate cyclase interactions. FASEB J 5: 2300-2303, 1991[Abstract/Free Full Text].

3.   Bachelet, M, Vincent D, Havet N, Marrash-Chahla R, Pradalier A, Dry J, and Vargaftif BB. Reduced responsiveness of adenylate cyclase in alveolar macrophages from patients with asthma. J Allergy Clin Immunol 88: 322-328, 1991[ISI][Medline].

4.   Bai, TR. Abnormalities in airway smooth muscle in fatal asthma: a comparison between trachea and bronchus. Am Rev Respir Dis 143: 441-443, 1991[ISI][Medline].

5.   Bayewitch, ML, Avidor-Reiss T, Levy R, Pfeuffer T, Nevo I, Simonds WF, and Vogel Z. Inhibition of adenylyl cyclase isoforms V and VI by various beta gamma subunits. FASEB J 12: 1019-1025, 1998[Abstract/Free Full Text].

6.   Cali, JJ, Zwaagstra JC, Mons N, Cooper DM, and Krupinski J. Type VIII adenylyl cyclase. A Ca2+ calmodulin-stimulated enzyme expressed in discrete regions of rat brain. J Biol Chem 269: 12190-12195, 1994[Abstract/Free Full Text].

7.   Codina, J, Hildebrandt J, Iyengar R, Birnbaumer L, Sekura RD, and Manclark CR. Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha beta heterodimer regulated by guanine nucleotide and magnesium. Proc Natl Acad Sci USA 80: 4276-4280, 1983[Abstract].

8.   Codina, J, Hildebrandt J, Sekura RD, Birnbaumer M, Bryan J, Manclark CR, Iyengar R, and Birnbaumer L. Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases. J Biol Chem 259: 5871-5886, 1984[Abstract/Free Full Text].

9.   Deery, WJ, and Rani CS. Protein kinase C activation mimics but does not mediate thyrotropin-induced desensitization of adenylyl cyclase in cultured dog thyroid cells. Endocrinology 128: 2967-2975, 1991[Abstract].

10.   Dooper, MWSM, Timmermans A, Aalbers R, deMonchy JGR, and Kauffman HF. Desensitization of the adenylyl cyclase system in peripheral blood mononuclear cells from patients with asthma three hours after allergen challenge. J Allergy Clin Immunol 92: 554-566, 1993.

11.   Emala, CW, Black C, Curry C, Levine MA, and Hirshman CA. Impaired beta -adrenergic receptor activation of adenylyl cyclase in airway smooth muscle in the basenji-greyhound dog model of airway hyperresponsiveness. Am J Respir Cell Mol Biol 8: 668-675, 1993[ISI][Medline].

12.   Hotta, K, Emala CW, and Hirshman CA. TNF-alpha upregulates Gialpha and Gqalpha protein expression and function in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 276: L405-L411, 1999[Abstract/Free Full Text].

13.   Iwami, G, Kawabe J, Ebina T, Cannon PJ, Homcy CJ, and Ishikawa Y. Regulation of adenylyl cyclase by protein kinase A. J Biol Chem 270: 12481-12484, 1995[Abstract/Free Full Text].

14.   Jacobowitz, O, and Iyengar R. Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2. Proc Natl Acad Sci USA 91: 10630-10634, 1994[Abstract/Free Full Text].

15.   Kawabe, J, Iwami G, Ebina T, Ohno S, Katada T, Ueda Y, Homcy CJ, and Ishikawa Y. Differential activation of adenylyl cyclase by protein kinase C isoenzymes. J Biol Chem 269: 16554-16558, 1994[Abstract/Free Full Text].

16.   Kitten, AM, Hymer TK, and Katz MS. Bidirectional modulation of parathyroid hormone-responsive adenylyl cyclase by protein kinase C. Am J Physiol Endocrinol Metab 266: E897-E904, 1994[Abstract/Free Full Text].

17.   Lai, HL, Yang TH, Messing RO, Ching YH, Lin SC, and Chern Y. Protein kinase C inhibits adenylyl cyclase type VI activity during desensitization of the A2a-adenosine receptor-mediated cAMP response. J Biol Chem 272: 4970-4977, 1997[Abstract/Free Full Text].

18.   Laurenza, A, and Seamon KB. High-affinity binding sites of [3H]forskolin. Methods Enzymol 195: 52-65, 1991[ISI][Medline].

19.   Lindeman, KS, Hirshman CA, Kuhl JS, Levitsky HI, and Emala CW. Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells. Biol Reprod 59: 1108-1115, 1998[Abstract/Free Full Text].

20.   Morris, DI, Robbins JD, Ruoho AE, Sutkowski EM, and Seamon KB. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter. J Biol Chem 266: 13377-13384, 1991[Abstract/Free Full Text].

21.   Pasquali, D, Rani CSS, and Deery WJ. Carbachol-induced decrease in thyroid cell adenylyl cyclase activity is independent of calcium and phosphodiesterase activation. Mol Pharmacol 41: 163-167, 1992[Abstract].

22.   Salomon, Y, Londos C, and Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541-548, 1974[ISI][Medline].

23.   Schears, G, Clancy J, Hirshman CA, and Emala CW. Chronic carbachol pretreatment decreases adenylyl cyclase activity in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 273: L640-L647, 1997[Abstract/Free Full Text].

24.   Scholich, K, Barbier AJ, Mullenix JB, and Patel TB. Characterization of soluble forms of nonchimeric type V adenylyl cyclases. Proc Natl Acad Sci USA 97: 2915-2920, 1997.

25.   Smith, PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85, 1985[ISI][Medline].

26.   Strittmatter, S, and Neer EJ. Properties of the separated catalytic and regulatory units of brain adenylate cyclase. Proc Natl Acad Sci USA 77: 6344-6348, 1980[Abstract].

27.   Tang, W-J, and Gilman AG. Type-specific regulation of adenylyl cyclase by G protein beta  subunits. Science 254: 1500-1503, 1991[ISI][Medline].

28.   Tang, W-J, Krupinski J, and Gilman AG. Expression and characterization of calmodulin-activated (type I) adenylyl cyclase. J Biol Chem 266: 8595-8603, 1991[Abstract/Free Full Text].

29.   Taussig, R, and Gilman AG. Mammalian membrane-bound adenylyl cyclases. J Biol Chem 270: 1-4, 1995[Free Full Text].

30.   Tolan, D, Conway AM, Pyne NJ, and Pyne S. Sphingosine prevents diacylglycerol signaling to mitogen-activated protein kinase in airway smooth muscle. Am J Physiol Cell Physiol 273: C928-C936, 1997[Abstract/Free Full Text].

31.   Yoshimura, M, and Cooper DM. Cloning and expression of a Ca2+-inhibitable adenylyl cyclase from NCB-20 cells. Proc Natl Acad Sci USA 89: 6716-6720, 1992[Abstract].

32.   Yoshimura, M, and Cooper DMF Type-specific stimulation of adenylyl cyclase by protein kinase C. J Biol Chem 268: 4604-4607, 1993[Abstract/Free Full Text].

33.   Yoshimura, M, Ikeda H, and Tabakoff B. µ-Opioid receptors inhibit dopamine stimulated activity of type V adenylyl cyclase but enhance the dopamine stimulated activity of type VII cyclase. Mol Pharmacol 50: 43-51, 1996[Abstract].

34.   Zimmermann, G, and Taussig R. Protein kinase C alters the responsiveness of adenylyl cyclases to G protein alpha  and beta gamma subunits. J Biol Chem 271: 27161-27166, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 279(4):C1008-C1015
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




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