Cyclic GMP regulates M₃AChR activity at plasma membranes from Airway smooth muscle

Figures & data

Table I. Agonist competition binding parameters of muscarinic receptors in tracheal smooth muscle plasma membranes.

	Additions
Agonist	MgCl2	MgCl2 plus GTPγS
Carbamylcholine
np-H	0.6 ± 0.1	1.0 ± 0.1
%RH	61.6 ± 6.3	n.d*
%RL	38.4 ± 6.1	100^§
pIC50H	7.3 ± 0.1	n.d*
pIC50L	5.3 ± 0.2	5.1 ± 0.1^†
Metacholine
np-H	0.7 ± 0.1	1.0 ± 0.1
%RH	52.1 ± 5.7	n.d*
%RL	47.9 ± 5.6	100^§
pIC50H	7.1 ± 0.1	n.d*
pIC50L	5.1 ± 0.1	5.1 ± 0.1^†
Oxotremorine
np-H	0.7 ± 0.1	0.9 ± 0.1
%RH	46.6 ± 5.7	n.d
%RL	53.4 ± 6.0	100^§
pIC50H	7.4 ± 0.2	n.d*
pIC50L	5.2 ± 0.1	5.2 ± 0.1^†

Tracheal smooth muscle plasma membranes were prepared as described in Methods. These membranes were used to perform competitive binding curves using [³H]QNB binding assays in a buffer containing 50 mM Tris-HCl pH 7.6, in presence of 5 mM MgCl₂ or 100 μM GTPγS plus 5 mM MgCl₂, which were analyzed to determine the interactions of the ligand with ‘one' or ‘two' independent classes of receptor binding sites. The proportion (% R_H, % R_L ± SE, high and low agonist site, respectively) and the affinity (pIC_50H, pIC_50L ± SE, high and low agonist binding site affinity respectively; n = 4) of the receptor sites were calculated as described in Methods. *Not detected; ^§one site receptor affinity; ^†pIC₅₀ of one binding site.

Table II. Antagonist competition binding parameters of muscarinic receptor in tracheal smooth muscle plasma membranes.

Antagonists	Additions
M2AChR selective	MgCl2	MgCl2+GTPγS
Methoctramine
np-H	1.0 ± 0.1	1.1 ± 0.1
pIC50 ^†	6.7 ± 0.1	6.5 ± 0.1
np-H	1.0 ± 0.1	1.0 ± 0.1
AF-DX 116
pIC50 ^†	5.7 ± 0.1	5.8 ± 0.1
M3AChR selective
4-DAMP
np-H	0.7 ± 0.1	1.0 ± 0.1
pIC50h	8.1 ± 0.2	n.d*
pIC50l	6.8 ± 0.6	6.9 ± 0.2

Native tracheal smooth muscle plasma membranes were isolated as described in Methods. Competitive binding curves using [³H]QNB binding assays were carried out in a buffer containing 50 mM Tris-HCl pH 7.6, in presence of 5 mM MgCl₂ or 100 μM GTPγS plus 5 mM MgCl₂ and analyzed to determine the interactions of the ligand with ‘one' or ‘two' independent classes of receptor binding sites. The pseudo-Hill coefficients (n_p-H) and the affinity (pIC_50h, pIC_50l ± SE, high and low antagonist binding site affinity respectively; n = 4) of the receptor sites were calculated as described in Methods. The values for the pIC₅₀ for the high affinity antagonist sites (pIC_50h) and low affinity antagonist sites (pIC_50l) were established and estimated. ^†pIC₅₀ of one binding site, *not detected.

Table III. Drugs competition binding parameters of muscarinic receptors in 4-DAMP alkylated plasma membranes from tracheal smooth muscle.

Muscarinic compounds	Additions
Antagonists	MgCl2	MgCl2 + GTPγS
Methoctramine
np-H	1.0 ± 0.1	1.1 ± 0.1
pIC50 ^†	6.4 ± 0.1	6.4 ± 0.1
np-H	1.0 ± 0.1	1.0 ± 0.1
AF-DX 116
pIC50 ^†	5.3 ± 0.1	5.3 ± 0.1
4-DAMP
np-H	1.0 ± 0.1	1.0 ± 0.1
pIC50 ^†	6.4 ± 0.6	6.4 ± 0.2
Agonists
Carbamylcholine
np-H	0.6 ± 0.1	1.0 ± 0.1
%RH	37.4 ± 4.5	n.d*
%RL	63.6 ± 5.0	100^§
pIC50H	7.3 ± 0.2	n.d*
pIC50L	5.1 ± 0.1	5.6 ± 0.2^†

4-DAMP mustard-alkylated tracheal smooth muscle plasma membranes were prepared as described in Methods. Muscarinic drug displacement assays were performed using similar quantities of active receptors. Competitive binding curves using [³H]QNB binding assays were carried out in a buffer containing 50 mM Tris-HCl pH 7.6, in the presence 5 mM MgCl₂ or 100 μM GTPγS plus 5 mM MgCl₂ and analyzed to determine the interactions of the ligand with ‘one' or ‘two' independent classes of receptor binding sites. The pseudo-Hill coefficients (n_p-H) and the affinity (pIC₅₀ ^† ± SE; n = 4) of the receptor sites were calculated as described in Methods. The proportion (% R_H, % R_L ± SE, high and low agonist site, respectively) and the affinity (pIC_50H, pIC_50L ± SE, high and low agonist binding site affinity, respectively; n = 4) of the receptor sites were calculated as described in Methods. *Not detected; ^§one site receptor affinity; ^†pIC₅₀ of one binding site.

Figure 1. Effect of cGMP in the presence or absence of ATP on the [³H]QNB binding from of plasma membranes from BTSM. Binding experiments were carried out in the presence of cGMP (○) and cGMP plus 5 mM ATP (•) and 1,500 pM [³H]QNB, 5 mM MgCl₂ and 2–4 µg of membrane proteins were assayed at 37°C as described in Methods. Specific [³H]QNB binding expressed as percentage of binding in the absence of nucleotides. The binding activity in the absence of cGMP was 1,100 ± 130 fmoles/mg protein and 1,450 ± 170 fmoles/mg protein in the presence of cGMP plus ATP. Each point represents the mean ± SE of four different membrane preparations assayed in triplicate.

Figure 2. Effect of PDE inhibitors on [³H]QNB binding in of plasma membranes from BTSM. Binding experiments were carried out at 37°C as described in Methods in the presence of 2–4 µg of membrane proteins, 1,500 pM [³H]QNB, 5 mM MgCl₂, 5 mM ATP and (•) increasing concentrations of cGMP or (▪) the specific inhibitor of PDE V, zaprinast (100 nM) or (○) the non-specific inhibitor of PDEs, IBMX (10 μM). Each point represents the mean ± SE of four different membrane preparations assayed in triplicate.

Figure 3. Kinetic curves of the binding of [³H]QNB in of plasma membranes from BTSM. Saturation concentration of [³H]QNB was 1,500 pM, 5 mM MgCl₂ and 2–4 µg of membrane proteins were assayed in the presence of IBMX (10 μM) at 37°C as described in Methods. Binding experiments were carried out in the presence of 10 nM cGMP (○), 5 mM ATP (▪) and 10 nM cGMP plus 5 mM ATP (•). Specific [³H]QNB binding expressed as fmoles/mg protein. Each point represents the mean ± SE of four different membrane preparations assayed by triplicate. *Statistical significant (p < 0.05). Insert shows the plot of the best line of fit through the first half-life of each curve from Figure 3, which is a method used to calculate the association rate constants (k_on) as described (Bennett and Yamamura 1985).

Table IV. Effects of nucleotides on the [³H]QNB binding parameters in tracheal smooth muscle plasma membranes.

Additions	Bmax (fmol/mg of protein)	KD (pM)	nH
cGMP	1209 ± 121	516 ± 79	1.2 ± 0.2
ATP	1369 ± 156	579 ± 47	1.3 ± 0.1
ATP plus cGMP	1721 ± 194	528 ± 57	1.9 ± 0.3

Tracheal smooth muscle plasma membranes were prepared as described in Methods. The [³H]QNB binding saturation experiments were carried out in a buffer containing 50 mM Tris-HCl pH 7.6, containing 5 mM MgCl₂ as described in Methods and the following conditions: 10 nM cGMP, 5 mM ATP and 10 nM cGMP plus 5 mM ATP. Results are the mean ± SE of four different membrane preparations assayed by triplicate. The values of B_max, K_D and n_H were calculated as described (Bennett and Yamamura 1985).

Table V. Effect of cGMP in the presence of ATP on the agonist and antagonist competition binding parameters of muscarinic receptors in tracheal smooth muscle plasma membranes.

Drug	Additions
	ATP	cGMP + ATP
Carbamylcholine
np-H	0.43 ± 0.08	0.87 ± 0.13
%RH	54.8 ± 0.05	n.d*
%RL	45.2 ± 0.06	100^§
pIC50H	7.21 ± 0.13	n.d*
pIC50L	5.01 ± 0.12	5.38 ± 0.06^†
4-DAMP
np-H	0.96 ± 0.08	0.64 ± 0.05
%RH	n.d*	28.4 ± 0.30
%RL	100^§	71.6 ± 0.70
pIC50H	n.d*	7.48 ± 0.10
pIC50L	6.55 ± 0.07^†	6.23 ± 0.02
Methoctramine
np-H	0.96 ± 0.01	0.99 ± 0.02
pIC50	6.52 ± 0.04	6.93 ± 0.07
Gallamine
np-H	0.99 ± 0.02	1.01 ± 0.03
pIC50	5.85 ± 0.11	5.42 ± 0.09
Atropine
np-H	1.07 ± 0.20	0.99 ± 0.11
pIC50	8.22 ± 0.06	8.18 ± 0.08

Tracheal smooth muscle plasma membranes were prepared as described in Methods. Competitive binding curves were carried out in a buffer containing 50 mM Tris-HCl pH 7.6, in presence of 5 mM MgCl₂ and 5 mM ATP or 10 nM cGMP. The experimental data were analyzed for ‘one' or ‘two' independent classes of receptor binding sites when appropriate. Thus, the proportion (% R_H, % R_L ± SE, high and low agonist site, respectively) and the affinity (pIC_50H, pIC_50L ± SE, high and low agonist binding site affinity, respectively; n = 4) of the receptor sites were calculated as described in Methods. *Not detected; ^§one site receptor affinity; ^†pIC₅₀ of one binding site.

Figure 4. Effect of cGMP on the [³H]QNB binding in Control plasma membranes and 4-DAMP mustard-alkylated plasma membranes. The [³H]QNB binding curves were perrformed as described in Methods for both native membranes (•) and 4-DAMP mustard-alkylated membranes (○) in the presence of 5 mM ATP, 5 mM MgCl₂ and increasing cGMP concentrations are indicated. Specific [³H]QNB binding is expressed as percentage of binding in the absence of nucleotides. Binding experiments were carried out at 1,500 pM [³H]QNB and 2–4 µg of membrane proteins were assayed at 37°C. The total binding activity was 1,570 ± 170 fmoles/mg protein in Control membranes and 549 ± 20 fmoles/mg protein in 4-DAMP mustard alkylated-membranes. Each point represents the mean ± SE of four different membrane preparations assayed in triplicate.

Figure 5. (A) Effect of cyclic nucleotide analogs (activator of PKG on the muscarinic activity of plasma membranes from BTSM. [³H]QNB binding in plasma membranes from BTSM was performed using 1,500 pM [³H]QNB and 2–3 µg of membrane proteins were assayed at 37°C as described in Methods. A titration of cGMP was performed in the presence of 5 mM ATP and 5 mM MgCl₂ under increasing concentration of Sp-8-pCPT-cGMPS were 0 (▪), 0.25 (•), 0.5 (□), 1.0 (○) and 5.0 μM (◊), respectively. (Insert: Activation curve obtained by plotting binding data obtained at 1 nM cGMP). (B) Effect of cyclic nucleotide analogs (inhibitor) of PKG on the muscarinic activity of plasma membranes from BTSM. [³H]QNB binding in plasma membranes from BTSM was performed using 1,500 pM [³H]QNB and 2–3 µg of membrane proteins were assayed at 37°C as described in Methods. A titration of cGMP was performed in the presence of 5 mM ATP and 5 mM MgCl₂ under increasing concentration of Rp-8-pCPT-cGMPS (Insert: Inhibition curve obtained by plotting binding data obtained at 10 nM cGMP). Sp-8-pCPT-cGMPS and Rp-8-pCPT-cGMPS concentrations were 0 (▪), 0.25 (•), 0.5 (□), 1.0 (○) and 5.0 μM (◊), respectively. Each point represents the mean ± SE of four different plasma membrane preparations assayed in triplicate.

Figure 6. Western blotting for PKG-II in P1 plasma membranes fractions from BTSM. Plasma membrane fraction (P₁) (20 µg) was subjected to 10% PAGE-SDS. Proteins were transfer to PDVF membranes and Western blot analysis with rabbit antisera directed against the PKG-II was performed. In addition, to identify the immunoreactivity proteins (positive bands), a Precision Plus protein^™ Western C^™ Pack from BioRAD and the enhanced chemiluminescence method as described by the manufacturer (Amersham ECL Western Blotting Reagent Pack from GE/Healthcare, Life Sciences) were used to identify the positive bands.

Figure 7. Autoradiograms of m2 and m3-immunoprecipitated ³²P-labeled polypeptides. Plasma membranes fraction was subjected to [³²P]phosphorylation using [γ³²P]ATP as described in Methods. Later, [³²P]-labeled membranes were solubilized using a detergent mixture containing 0.1% Digitonin-0.02% sodium cholate and incubated with specific anti m3 or anti m2AChR antibodies as described in Methods. Immunoprecipitates were collected with protein A/G-agarose beads by low speed centrifugation an incubated with cracking SDS solution and subjected to 10% SDS-PAGE. Gels were dried and exposed during 7–12 days at −80°C under X-film (Hyperfilm®). ³²P-labeled m3-AChR immunoprecipitated polypeptides in the absence (lane A) or presence (lane B) of 10 nM cGMP and ³²P-labeled m2-AChR immunoprecipitated polypeptides in the absence (lane C) or presence (lane D) of 10 nM cGMP. Arrows indicates the molecular masses m₃AChR of 67 KDa and 52 KDa for m₂AChR. All ³²P-labeled phosphoproteins were adjusted to the same level for comparison.

Figure 8. Proposed model of molecular regulation of M₃AChR induced by PKG-II phosphorylation at plasma membrane from BTSM. All molecular compnents are membrane bound entities. (A) Muscarinic agonist (Acetylcholine) binds to M₃AChR and activates the heterotrimeric Gq₁₆, which stimulates the NPR-GC and increases the cGMP production. (B) Cyclic GMP activates the PKG-II and produces M₃AChR phosphorylation inducing a conformation changes to produce M₃AChR dimer, which stabilizes or ‘freezes' the M₃AChR population, in a refractory state to agonist activation, and prone to antagonist binding, which helps to understand the molecular mechanisms of muscarinic antagonist drug action.

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