Muscarinic receptor subtypes in feline tracheal submucosal gland secretion H. ISHIHARA, S. SHIMURA, M. SATOH, T. MASUDA, H. NONAKA, H. KASE, T. SASAKI, H. SASAKI, T. TAKISHIMA, AND K. TAMURA First Department of Internal Medicine, Tohoku University School of Medicine, Sendai; Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo, Shizuoka; and Second Department of Internal Medicine, Yamanashi Medical College, Kofu, Japan Ishihara, I$, S. Shimura, M. Satoh, T. Masuda, H. Nonaka, H. Kase, T. Sasaki, H. Sasaki, T. Takishima, and K. Tamura. Mucarinic receptor subtypes in feline tracheal submucosal gland secretion. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L223-L228, 1992.-To determine what muscarinic receptor subtype regulates [ Ca2+]; mediating airway submucosal gland secretion, we examined the effects of atropine (Atr), pirenzepine (PZ), ll( [2-(diethylamino)methyl-l-piperidinyl]acetyl)-5,11-dihydro-GH-pyrido (2,3-b)(I,4)-benzo-diazepin-6-one (AF-DXll6) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) on methacholine (MCh)evoked [Ca2+]i rise in acinar cells, and compared this with mucus glycoprotein (MGP) and electrolyte secretion evoked by MCh from submucosal glands isolated from feline trachea. [Ca”+]; was measured with the Ca2+-sensitive fluorescent dye, fura 2. We determined MGP secretion by measuring TCAprecipitable 3H-labeled glycoconjugates and electrolyte secretion by the change in the rate constant of 22Na-efflux from isolated glands. Half-maximal inhibitory concentrations (I&J of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked [Ca2+]; rise were low7 M, 6 X lOA M, 8 X lo-’ M, and 6 X IO-’ M, respectively. I& of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked MGP secretion were lOA M, 2 X 10B5 M, 8 x IO-’ M, and 6 X lo-’ M, respectively. MCh ( low5 M)evoked 22Na efflux was significantly inhibited by 10V7 M 4DAMP and 10B7 M Atr (P < 0.01, each) but not by low7 M PZ. Receptor binding assays with [“HI quinuclidinyl benzilate showed that the K; values for PZ, AF-D X 116, 4-DAMP and Atr were 2.2 X lo-’ M, 6.6 X 10V7 M, 6.2 X 10-l’ M, and 2.9 X 10 -lo M, respectively. These findings suggest that cholinergic stimulation induces MGP and electrolyte secretions by the activation of muscarinic M3 receptors, involving [Ca2+]; as the second messenger in the secretory cells of airway submucosal glands. cat; mucus glycoprotein secretion; electrolyte secretion OF THE DEVELOPMENT Of SeleCtive antagonists, muscarinic receptor subtypes have now been identified in several tissues (8), and advances in molecular biology have confirmed their existence. As many as five different receptor subtypes have now been cloned and identified (4). Muscarinic receptors are morphologically shown to be present on airway gland cells (2, 3), and muscarinic stimulation is the most potent stimulant of airway submucosal gland secretion (21, 23). Recently muscarinic receptors of airways have been pharmacologically subclassified into three subtypes; M1, M2, and M3 receptors (1). There have been few reports concerning the role of muscarinic receptor subtypes in airway submucosal gland secretion. Autoradiographic investigations of human lungs have demonstrated the coexistence of M1 (36%) and M3 (64%) receptors over airway submucosal glands (18). Yang et al. (29,30) have found the presence of both M, (27%) and MS (73%) receptors on isolated submucosal BECAUSE
1040-0605/92
$2.00
Copyright
0 1992
gland cells of swine trachea and speculated that electrolyte and/or water secretion is stimulated via activation of M1 receptors and mucin secretion is stimulated via activation of M3 receptors. Meanwhile, Gater et al. (11) have reported that the muscarinic receptor subtype mediating mucus secretion in cat trachea has an intermediate affinity for pirenzepine, suggesting an “intermediate” between M1 and M2 receptor subtypes. We have shown that muscarinic stimulation produces a rise in intracellular calcium ion concentration ( [ Ca’+]J, resulting in mucus glycoprotein secretion (14) and electrolyte secretion (20). However, to the best of our knowledge, there have been no reports on the kind of muscarinic receptor subtype that mediates [Ca2+]i in airway submucosal gland cells. Therefore, we examined the effects of Ml-, M2-, and M3-receptor antagonists on [Ca2+]; rise by cholinergic stimulation using isolated feline tracheal submucosal glands, which enables us to examine submucosal gland secretion in a well-defined condition by excluding possible effects from surrounding tissues (2 1). MATERIALS
AND
METHODS
We measured methacholine (MCh)-induced [Ca”+]; rise in the presence and absence of each of four muscarinic receptor antagonists: atropine, pirenzepine (PZ), 11( [ 2- (diethylamino)methyl-l-piperidinyl]acetyl)-5,11-dihydro-6H-pyreido(2,3-b)(1,4)-b enzo-diazepin-6-one (AF-DXllG), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP methiodide) and compared this with radiolabeled glycoconjugate secretion and 22Na efflux from isolated feline tracheal submucosal glands by the methods described in our previous reports (2025). Trichloroacetic acid (TCA) -precipitable glycoconjugate secretion represents mucus glycoprotein (MGP) secretion (24)) and 22Na efflux represents electrolyte and/or fluid secretion from submucosal glands (20). Isolation of submucosal glands from feline trachea. Submucosal glands were isolated from feline tracheae by the method previously reported by us (26). The tracheae were removed from 34 cats (2.3-3.9 kg body w-t) under light anesthesia with intramuscular ketamine hydrochloride and intravenous thiopental sodium (30 mg/kg). Immediately after removal, submucosal glands were isolated under a stereoscopic microscope with two pairs of sharpened tweezers and microscissors. To avoid tissue damage during isolation, care was taken to isolate the submucosal gland by picking up some of the connective tissue attached to the glands. The entire procedure was performed in a modified Krebs-Ringer bicarbonate (KRB) solution containing the following (in mM): 125 NaCl, 5 KCl, 1.2 Mg CL, 1 CaC12, 25 NaHC03, 1.2 NaHP04, and 11 glucose with bubbling of 5% C02-95% O2 and pH 7.4 at 20°C. Measurement of 3H-labeled glycoconjugate secretion. The isolated glands were incubated with 2 ml of medium 199 containing D [6-“H] glucosamine hydrochloride (2.5 &i/ml, sp act 27-40 the American
Physiological
Society
L223
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L224
MUSCARINIC
RECEPTORS,
[CA2+]i
IN
Ci/mmol; Amersham, Tokyo) for 16 h and then allowed to incubate further at 37°C for two successive Z-h periods (period I and period II) in 2 ml of culture media without a radiolabeled precursor. Each antagonist was added to the media 15 min before the end of period I. In period II, culture was done in the presence of MCh (10v5 M) and each muscarinic antagonist. At the end of each period, the culture media were harvested and precipitated with 10% TCA and 1% phosphotungstic acid (PTA) at 4°C overnight. The precipitated radioactivity was measured in a liquid scintillation counter (LSC-1000, Aloka, Tokyo). The ratio of precipitated radioactivity [disintegrations/ min (dpm)] from period II to that from period I was determined for each sample and was termed the secretory index. The effect of antagonists was determined by comparing the secretory indexes of the antagonist-treated samples with those of matched control samples. Measurement of [Ca2”/i. [Ca”‘]; in the isolated glands was determined by the fura 2 method with a microscopic system CAM-220 (Japan Spectroscopic, Tokyo). The isolated glands were incubated at room temperature for 2-4 h in the loading solution, which contained 5 PM of the acetoxymethyl ester of fura 2 (fura 2/AM) and 0.2% of a detergent, Cremophor EL, to increase the solubility of fura 2/AM. After fura 2 loading, the glands were further incubated in fura 2-free KRB. Each gland was then put on a cover glass (0.12-0.17 mm of thickness) on the stage of an inverted microscope, which was warmed to 37°C by tungsten electrical coils. The microscopic field was adjusted to the acinar portion of the glands with a magnification of x400. The excitation wavelengths were 340 and 380 nm, and the emission wavelength was 500 nm. The fluorescence ratio was recorded as a function of time and converted to [Ca2+]; by the method of Grynkiewicz et al. (13) and Scanlon et al. (22) by the equation [Ca2+]i = (R - R,i,)/(R,,, - R) &* S where R is the experimentally determined fura 2 fluorescence ratio, and the dissociation constant (&) is assumed to be 224 nM (13). R,,, and Rmin were determined by the addition of 10s5 M ionomycin and 5 mM ethyleneglycol-bis(@-aminoethyl ether)N,N,N’,N’ tetraacetic acid (EGTA), respectively. The constant S is the ratio of fluorescence at 380 nm in a Ca2+-free solution to that in a Ca2+-containing solution in the presence of ionomycin. Measurement of 22Na efflux. Ten to twenty submucosal glands were incubated in 5% C02-95% O2 bubbled KRB containing 5 &i/m01 of 22NaC1 (sp act 502.6 mCi/mg, New England Nuclear, Boston, MA) at 37°C for 2 h as in our previous report (20). 22Na-loaded glands were transferred to a perfusion apparatus of a vertical acryl resin tube. The perfusate, a KRB solution gassed at 37°C either with or without drugs, was continuously pumped to the glands at a constant flow rate, and samples of effluent were collected at 18-s intervals over a period of 10 min. The 22Na radioactivity in collected effluent samples and that remaining in the gland samples at the end of the experiments were determined with an auto-gamma counter (ARC-500, Aloka, Tokyo). Drugs were applied to the glands by changing the perfusate to one containing a defined concentration of the drug required. The instantaneous rate constant for each individual sampling period was calculated as described previously (20). Stimulation with MCh was performed in the 22Na-loaded glands after 5 min of perfusion. The change in the instantaneous rate constant with MCh was estimated by comparing the baseline rate constant (mean of the rate constants during 3-5 min) with that of the maximal response in the presence or absence of each antagonist. Receptor binding assays. Two hundred fifty isolated glands from nine animals were used for the receptor-binding assays. Isolated glands were kept at -80°C until use. For membrane preparation, the feline tracheal submucosal glands were homogenized in ice-cold 50 mM Na-K phosphate
TRACHEAL
SUBMUCOSAL
GLANDS
buffer, pH 7.4 (40 mM K2HP04, 10 mM NaH2P04) with a Polytron homogenizer (setting 5) for three times (20 s each). The homogenate was subjected to a low-speed centrifugation at 1,200 g for 5 min. The supernatant was further centrifuged at 35,500 g for 15 min at 4°C. The final pellet was resuspended in the same buffer to a concentration of 8-10 pg protein/ml. The homogenate was used immediately for the receptor-binding assays. Protein was determined by the method of Lowry et al. W) For the radioligand binding assay, the membrane protein (810 pg) was added to the reaction mixture containing increasing concentrations of [3H] quinuclidinyl benzilate ( [ 3H] QNB, 50 Ci/ mmol; Amersham, Tokyo, Japan) in the 50 mM Na-K phosphate buffer (pH 7.4) to a total volume of 1.1 ml. Nonspecific binding was determined by the addition of I PM atropine. Samples were incubated at 25°C for 2 h, and binding was terminated by filtering the samples over Whatman GF/C glass fiber filters with the use of a Brandel cell harvester apparatus with three 5-ml washes of ice-cold buffer. The filters were placed in scintillation vials with Scintisol EX-H (Wako Pure Chemicals, Osaka, Japan), and radioactivity was counted by a Packard 2200CA scintillation counter (Packard Instruments). Competition assays were done as described above except that 1 nM [“H]QNB was added and various concentrations of a specific competitor were introduced, making a final volume of 1.1 ml. Reagents. Drugs used in this study were fura 2/AM (Dojin, Kumamoto, Japan), Cremophor EL (Nakarai Chemicals, Kyoto, Japan), ionomycin (Calbiochem, La Jolla, CA), acetyl,&methylcholine hydrochloride (methacholine), EGTA (Wako Pure Chemicals, Osaka, Japan), atropine sulfate, pirenzepine dihydrochloride (Sigma Chemical, St. Louis, MO), medium 199 (Flow Laboratory, McLean, VA), and Aquasol 2 (New England Nuclear, Boston, MA). AF-DXll6 was a generous gift from Boehringer-Ingelheim, Ingelheim, FRG, and 4-DAMP was a generous gift from Dr. R. Barlow, University of Bristol, Bristol UK. Statistical analysis. IC 5. values and Hill coefficients were determined from computerization of the logit log curve. The Cheng-Prusoff equation (5) was used to calculate inhibitor constant (Ki) values from the ICso values. & and the maximum binding capacity (B,,,) were determined by the nonlinear multipurpose curve-fitting program LIGAND (19). Data are means t SE. For multiple mean comparisons, analysis of variance and Duncan’s multiple range test were used. For mean comparison, the two-tailed paired or unpaired Student’s t test was used, and the Cochran Cox t test was used when Bartlet’s test for uniformity of variance showed it to be nonuniform. P < 0.05 was considered statistically significant. RESULTS
[Ccz’+/i rise. Ninety-five percent or more of the isolated glands responded significantly to MCh and produced significant rises in [Ca’+];. After stimulation with 10m5 M MCh, [ Ca2+]i increased from the resting level of 135 nM to 380 nM, and the increase was 344 t 36% (n = 26) of the resting level when expressed as a percentage of the peak value after stimulation to that of the resting level of each sample. After we confirmed the significant rises in [Ca2+]i in response to 10v5 M in control isolated glands, the other isolated glands from the same animals were used for the antagonist experiments. Each antagonist at 10-10-10-5 M was added to the KRB solution containing fura T&loaded isolated glands 15 min before each measurement of [Ca2+]; in response to 10V5M MCh.
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MUSCARINIC
RECEPTORS,
[CA2+]i
IN
The inhibitory effect of the antagonist was obtained by comparing the response to 10m5 M MCh in the presence of the antagonist with that to 10B5 M MCh in the absence of antagonist (control). The rise in [ Ca2+]; with MCh in the presence of each antagonist at 10-10-10-5 M was divided by that with MCh in the absence of any antagonist, and the inhibition due to antagonist was expressed as a percentage of the latter. Inhibition curves for four muscarinic antagonists on the MCh-induced [Ca”‘]i rise are summarized in Fig. 1 and Table 1. 4-DAMP produced an inhibitory curve similar to that for atropine, as shown in Fig. 1, and ICSo values for $-DAMP and atropine were 8 x IO-’ and 6 x lo-’ M, respectively. PZ and AF-DX116 yielded ICSo values of 10m7 and 6 X 10D6 M, respectively, and PZ at 10m6 M produced a significantly stronger inhibition than did AF-DX116 (P < 0.05) (Fig. 1 and Table 1). Thus 4DAMP was 100-fold more potent than PZ and l,OOO-fold [Ca’] (%)
’ rise
PO-
0
20 -
OI -;or+l
I
I -7 of antagonist
-8 Concentration
I
I
-6
-5
(log M >
Fig. 1. Inhibition curves of low5 M methacholine (MCh)-evoked intracellular calcium ion concentration ([ Ca2+]J rise for 4 muscarinic receptor antagonists: pirenzepine (o), 11{[2-(diethylamino)methyl-l-piperidinyllacetylj-5, ll-dihydro-GH-pyrido(2,3-b)(1,4)-benzo-diazepin-6one (AF-DXllG), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, w), and atropine (A). * P < 0.05, difference between 4-DAMP and pirenzepine. “f P < 0.05, difference between pirenzepine and AFDXl16. Results are means for 3-12 experiments.
TRACHEAL
SUBMUCOSAL
L225
GLANDS
more potent than AF-DX116 in antagonistic action. To understand the effects of PZ and $-DAMP on Ca2+ release from intracellular storage due to muscarinic receptor stimulation, we examined the effects of 10D7M PZ and 10B7M 4-DAMP on [Ca”‘]i rise in response to 10m5M MCh in a Ca2+-free solution. For Ca2+ depletion, 4 mM EGTA was added to the Ca2+-depleted solution, which was prepared by removing CaC12from the KRB solution. 4-DAMP (10m7M) abolished the MCh-evoked [Ca”‘]; rise (2 t 2% of control, P < 0.001, n = 4), whereas PZ (10m7M) did not significantly alter the MCh-evoked [Ca2+]i rise (122 t 40% of control, n = 5) in the Ca2+free condition. MGP secretion. The inhibitory effect of each antagonist on [3H]glycoconjugate secretion was obtained by comparing the increase in response to low5 M MCh in the presence of the antagonist with that in the absence of the antagonist, as described above. Inhibition curves for four muscarinic receptor antagonists on MCh (10S5 M) -induced MGP secretion are summarized in Fig. 2 and Table 2. $-DAMP has an inhibitory effect as potent as that of atropine, and IC50 values for 4-DAMP and atropine were 8 X lo-’ and 6 X lo-’ M, respectively. IC50 values for PZ and AF-DX116 were 10V6 and 2 X low5 M, respectively. 4-DAMP had a IOO-fold more potent inhibitory effect than did PZ and a l,OOO-fold more potent inhibitory effect than did AFDX116. Furthermore, PZ at 10B5 M produced significantly stronger inhibition than did AD-DX116 (P < 0.05, Fig. 2 and Table 2). 22Na efflux. Because the most dominant difference in antagonistic action in MCh-evoked [Ca2+]; rise or MChevoked MGP secretion between PZ and $-DAMP was observed at the concentration of low7 M of each antagonist, we examined the effects of 10m7M PZ and 10D7M 4-DAMP on MCh (low5 M)-induced 22Na efflux. Rate constants of 22Na efflux evoked by low5 M MCh were completely blocked by low7 M atropine, as shown in Fig. 3. PZ (10m7M) tended to reduce MCh-evoked 22Na-efflux from 349% of baseline rate constant (MCh alone) to 238% of baseline (MCh plus PZ), but the difference was not statistically significant. $-DAMP showed a significant inhibitory action and inhibited 22Naefflux to 130% of baseline rate constant, which was significantly lower than that with MCh alone (P < 0.01) or by PZ (P c 0.05, Fig. 3). Furthermore, 10B7 M atropine exhibited significantly lower rate constants than did 4-DAMP (P < 0.05, Fig. 3).
Table 1. Effects of each muscarinic antagonist on [Ca2+]i evoked by low5 M methacholine in isolated feline tracheal submucosal glands Antagonist
Pirenzepine AF-DXll6 4-DAMP Atropine
92.Okl9.7 95.3tl5.1 112.3kl6.0 89.2227.4
(6) (5) (5) (4)
96.2kl2.7 88.9kl7.2 81.6t25.3 82.4t17.3
(5) (5) (3) (7)
113.8tl7.1 82.8t12.7 37.ltl4.6* 20.5t8.3
Concentration,
(3) (5) (4) (8)
Data are means t SE expressed as % of controls (methacholine alone); values (diethylamino)methyl-l-piperidinyl]acetyl)-5,ll-dihydro-6H-pyrido(2,3-b)(l,4)-benzo-diazepin-6-one; * P < 0.05 compared with pirenzepine, t P < 0.05 compared peridine methiodide.
M
55.2kl7.6 88.7219.8 3.3&1.2* 8.9k2.6 in parentheses with
(12) (5) (6) (5)
15.8+2.5j76.1t19.0 9.0t5.4 1.5t0.6
(11) (6) (8) (5)
l.Ot3.6 40.7t18.0 -0.5t2.7 0.6tl.3
are numbers of experiments. AF-DXll6, 4-DAMP, 4-diphenylacetoxy-N-methylpi-
(5) (6) (4) (5) 11( [2-
AF-DXll6.
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L226 MGP (%>
MUSCARINIC
RECEPTORS,
[CA2+]i
IN
secretion I
60
1
n
0’ h--,-1
I
1
-10
-9
-8
Concentration
I
I
-7
-6
of antagonist
(log
---_A I
-5 M>
Fig. 2. Inhibition curves of 10s5 M MCh-evoked (TCA)-precipitable [“HI glycoconjugate secretion for 4 muscarinic receptor antagonists: pirenzepine, AF-DXll6,4-DAMP, and atropine. Symbols are same as in Fig. 1. * P < 0.05, ** P < 0.01, difference between 4-DAMP and pirenzepine. t P < 0.05, difference between pirenzepine and AF-DXll6. Results are means for 3-11 experiments. MGP, mucus glycoprotein.
Receptor-binding assays. [3H]QNB binding to submucosal gland homogenates was concentration and time dependent, and the specific binding was 82%. [3H]QNB saturation curves yielded a linear Scatchard plot. The & value was 67 t 7.2 pM, and B,,, was 1,500 t 62 fmol/ mg protein (n = 3). The Kd value was similar to those reported in rat cerebral cortex (42 pM from Ref. 28 and 21 pM from Ref. 27) and rat heart (21 pM from Ref. 27). These results indicate that the present receptor binding assays enable us to estimate the specific binding of [3H]QNB to the muscarinic receptors in submucosal gland cells. Ki and Hill coefficients of four different muscarinic antagonists are shown in Table 3. The Ki value of 4DAMP was similar to that of atropine and was 10m2and lOa or lessthan that of PZ and AF-DX116, respectively. DISCUSSION
The degree of inhibition by a receptor antagonist may be related to the concentration of a receptor agonist, and it is important to assessthe action of antagonists as well
TRACHEAL
SUBMUCOSAL
GLANDS
as agonists over a wide range of doses. However, our previous study (14) revealed an inhibitory action of fura 2 on mucus secretion from submucosal glands, which is dominant in the stimulation by agonists at lower concentrations. This makes it difficult to examine the shifts of the dose-response curves in [Ca2+]i of acinar cells of isolated glands and to obtain mean effective concentration (EC& of pA2 of each antagonist whereas no significant inhibitory action of fura 2 is observed when stimulated by MCh at higher concentrations (10D5 or 10W4 M). Therefore, we adopted the comparison of IC50 of each antagonist over a wide range of concentrations on [Ca2+]i rise by MCh at a relatively higher concentration of 10D5M in the present study. In this study we demonstrated that 4-DAMP, an M3receptor antagonist, is loo-fold more potent than PZ, an M1 receptor antagonist, and is l,OOO-fold more potent than AF-DXll6, an M2-receptor antagonist, in inhibiting [Ca2+]i rise by MCh in acinar cells of isolated glands. Furthermore, $-DAMP produced inhibitory curves similar to atropine, a nonspecific muscarinic receptor antagonist. These findings indicate that muscarinic agonists stimulate [Ca2+]i rise by activating Ma-muscarinic receptor subtypes in airway submucosal gland cells. Our previous report (14) indicated that MCh, especially at higher doses, produces [Ca2+]i rise both by release from intracellular storage and influx from the extracellular medium. In the present study, $-DAMP abolished a transient [Ca2+]; rise in a Ca2+-free solution, whereas PZ did not. This finding indicates that both the Ca2+ influx and its release from intracellular storage are coupled with the stimulation of MS-muscarinic receptor subtypes. It has been demonstrated that muscarinic receptor stimulation results in the breakdown of membrane phosphatidylinositol 4,5-bisphosphate and the formation of inositol 1,4,5trisphosphate (IP3) and that IP3 stimulates Ca2+ release from intracellular storage and, in the presence of inositol 1,3,4,&tetrakisphosphate (IP4), induces Ca2+ influx from the extracellular medium in other exocrine gland cells (10). The present findings provide evidence for phosphoinositide turnover in the coupling between M3-muscarinic receptor stimulation and mucus secretion in feline tracheal submucosal gland cells, similar to that in other exocrine gland cells. Our previous experiment (14) indicated that muscarinic receptor stimulation induces MGP secretion mediating the intracellular Ca2+system as a second messenger in feline tracheal submucosal glands. 4-DAMP showed the most potent inhibitory action to MCh-evoked glycoconjugate secretion among the three antagonists, an
Table 2. Effects of each muscarinic antagonist on trichloroacetic acid-precipitable [3H]glycoconjugate secretion evoked by 10B5M methacholine from isolated feline tracheal submucosal glands Antagonist lo-l0
Pirenzepine AF-DXll6 4-DAMP Atropine with
1225224.9 77.8kl9.4 81.6kl6.4 114.3kl5.5
1o-g
(5) (5) (5) (6)
102.lt24.8 8l.Otl0.8 99.lk21.5 128.2tl3.2
Data are mean % controls (methacholine pirenzepine, $ P < 0.05 compared with
1o-8
(5) (11) (5) (4)
110.3tl6.0 82.0t14.6 25.lt4.2* 38.2kl5.2
alone) f: SE; values AF-DXll6.
Concentration,
M 1o-7
(10) (4) (3) (9)
in parentheses
116.9tl9.3 86.2t17.6 11.2&9.3? 0.75kl2.1 are numbers
lo+
(10) (4) (4) (4)
1o-5
68.1t7.9 76.0t21.8 -7.7+1.3”r -14.6kl.7
of experiments.
(10) (5) (4) (3) *
16.3+7.5$ 55.9kl5.3 1.3kl.8 -14.3t4.3
(4) (11) (4) (6)
P < 0.05, “f P < 0.01 compared
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MUSCARINIC
RECEPTORS,
[CA2+]i
IN
TRACHEAL
SUBMUCOSAL
L227
GLANDS
glands. Autoradiographically, the coexistence of M1 and MS receptors over human airway submucosal glands has 400been demonstrated by Mak et al. (18). Yang et al. (30) have reported that acetylcholine caused a Ca2+-dependent decline in intracellular 36C1of submucosal gland cells 051 rP< of diisopropylfluorophosphate-treated swine trachea, 300and this loss of intracellular Cl was completely blocked by PZ or atropine. From these observations, they speculated that electrolyte (or fluid) secretion from airway 200 submucosal glands is coupled mainly with M1 receptor stimulation. In contrast, our present study shows the main role of M3 receptors in electrolyte secretion from feline tracheal submucosal glands. The difference belootween the results of Yang et al. (30) and our own may be due to differences in the species used for experiment. Another possible explanation is that Yang et al. used a single and relatively higher concentration of PZ in their OMCh MCh MCh MCh experiment and also did not examine the effects of M3 alone t t t receptor antagonist. Although PZ is considered to be a PZ 4 DAMP ATR selective antagonist for M1 receptors, the selectivity is Fig. 3. MCh (lo-“)-evoked 22Na efflux from isolated glands in absence lost at higher doses and it acts as an antagonist for MB or presence of each antagonist [10m7 M pirenzepine (PZ); 10v7 M 4receptors in the heart and for M3 receptors in smooth DAMP, or 10s7 M atropine (Atr)]. Results are expressed as % of muscle (8, 13, 17). For example, Gunst et al. (12) have baseline rate constant and means & SE for 3-5 experiments. ** P < reported that 10m6M PZ antagonizes M3 receptors of 0.05, compared with MCh alone. canine tracheal smooth muscle, whereas 10m8M PZ does action similar to the [Ca2+]i rise by MCh, in the present not. Our present experiment also demonstrates signifistudy. TCA-precipitable glycoconjugates secreted from cant inhibition by PZ at 10V5 and/or 10D6 M of both isolated glands represent MGP (24). Therefore, the pres- [Ca2+]i rise and MGP secretion, compared with that by ent finding indicates that MGP secretion evoked by AF-DXll6, as shown in Figs. 1 and 2. Both Mak et al. muscarinic stimulation is mediated by an MS-subtype (18) and Yang et al. (30) adopted a single concentration muscarinic receptor. of a relatively higher dose (low6 M) for their experiments. Previous reports, including our own (6, 15, ZO), have M3 receptors in airway glandular cells may be more shown that submucosal glands secrete not only MGP sensitive to PZ than those in smooth muscles. (mucin) but also electrolyte with fluid. Furthermore, the The present experiments including receptor-binding latter is detected by determining 22Na efflux from iso- assays indicate few or no M2 receptors. For example, the lated submucosal glands of feline trachea, which repre- present receptor-binding assays showed that the Ki value sents Na-K pump activity of the secretory cells (20). of AF-DXll6 was 660 nM (Table 3), which is much Although a limited experiment, 4-DAMP was the most larger than Ki values reported in the tissues possessing potent in inhibitory action on 22Na efflux from isolated M2 receptors. Watson et al. (27) reported that the K; feline submucosal glands. This indicates that electrolyte value of AF-DXll6 in rat heart is 39 nM, determined by (or fluid) secretion is also mediated mainly by MS-mus- [3H]QNB binding assay. After the submission of this carinic receptor subtypes in the submucosal gland. Meanmanuscript, Dorge et al. (7) reported that 4-DAMP has while, the inhibition by PZ was smaller than that by the an antagonistic action to M1 receptors in a degree similar control (MCh alone), and inhibition by 4-DAMP was to the action to M3 receptors. Our receptor binding assay significantly smaller than that of Atr. As well, PZ seems showed that the Hill coefficient value of PZ was 0.71, to have a stronger inhibitory action on [Ca2+]i rise than suggesting two or more populations of the binding recepthat on MGP secretion by MCh, as seen in Figs. 1 and tors. It is also possible that M1 and M3 receptors coexist 2. These findings do not eliminate the possibility that an in submucosal gland cells as indicated by previous reports M1 receptor has a small role in the electrolyte secretion (11, 18, 29). However, ID50 values for PZ in glycoconjufrom airway submucosal glands and that it may be too gate secretion and [Ca2+]i rise are 10D6 and 10m7M, small to detect by the present experimental design. respectively. These values are much larger than 1.8 X Some investigators have suggested the presence of M1 IO-’ M, which is reported as an apparent & value of M1 receptor in addition to M3 receptor in airway submucosal receptors for PZ (9). Therefore, M3 receptors functionally **Na-efflux (Oh of
control
)
Table 3. Dissociation constants and Hill coefficients of different muscarinic antagonists for muscarinic receptor subtypes Antagonist
Dissociation constants, Hill coefficients, nH
nM
Dissociation constants were derived from are means t SE of 3 experiments performed
Pirenzepine
AF-DX116
22t2.6 0.71~0.003
660t92 1.03kO.12
4DAMP
Atropine
0.62kO.089 0.65t0.05
0.29t0.044 0.96t0.07
the experimental values for half-maximal inhibitory concentration in duplicate. For each curve, 6-9 concentrations were used.
as described
in METHODS.
Values
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L228
MUSCARINIC
RECEPTORS,
[CA2+]i
IN TRACHEAL
SUBMUCOSAL
GLANDS
play a primary role in mucus secretion and [ Ca2+]i rise in feline tracheal submucosal glands. In conclusion, in the present study, we demonstrated that muscarinic stimulation induces MB-receptor-operated [Ca”‘]i rise and MGP and electrolyte (or fluid) secretion from the secreting cells of feline tracheal submucosal glands. These findings are important in the development of effective therapeutic methods for mucus hypersecretion in various pulmonary diseases.
receptor reserve and P-adrenergic sensitivity in tracheal smooth muscle. J. Appl. Physiol. 67: 1294-1298, 1989. 13. Grynkiewicz, G., M. Poenie, and R. Y. Tien. A new generation of Ca2+ indicators with greatly improved fluorescence properties.
We gratefully acknowledge Professor Akinori Nishiyama and Dr. Yoshitaka Saito at the Department of Physiology, Tohoku University School of Medicine, for their helpful advice and discussions, Dr. Ronald Scott and Elizabeth Crittenden for correcting the English, and Kumiko Shibuya for typing the manuscript. This study was supported by Scientific Grant 01570422 from the Ministry of Education, Science, and Culture of Japan. Address for reprint requests: T. Takishima, First Department of Internal Medicine, Tohoku University School of Medicine, l-l Seiryomachi, Aoba-ku, Sendai 980, Japan.
Appl. Physiol. 56: 426-430, 1984. 16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193: 265-275,195l. 17. Maclagen, J., and D. Faulker. Effect of pirenzepine and galla-
Received 24 October 1990; accepted in final form 9 September 1991. REFERENCES P. J. Muscarinic receptor subtypes: implications disease. Thorax 44: 161-167, 1989.
1. Barnes, 2. Barnes, baum.
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Roberts,
and
for lung
C. B. Bas-
Muscarinic receptors in lung and trachea: autoradiographic localization using [3H] quinuclidinyl benzilate. Eur. J. Pharmucol.
86: 103-106,1983. 3. Basbaum, C. B., M.
A. Grillo, and J. H. Widdicombe. Muscarinic receptors: evidence for nonuniform distribution in tracheal smooth muscle and exocrine glands. J. Neurosci. 4: 508-520, 1984.
4. Bonner,
T. I., N. J. Buckley,
A. C. Young,
and
M. R. Brann.
Identification of a family of muscarinic acetylcholine receptor genes. Science Wash. DC 236: 600-605, 1987. 5. Cheng, Y. C., and H. R. Prusoff. Relationship between the inhibition constant (K;) and the concentration of inhibitor which causes 50% inhibition (I&) of an enzymatic reaction. Biochem. PhurmacoZ. 6. Corrales,
22: 3099-3108,1973. R. J., J. A. Nadel,
and J. H. Widdicombe. Source of the fluid component of secretions from tracheal submucosal glands in cats. J. Appl. Physiol. 56: 1076-1082, 1984.
7. Dorge, F., J. Wess, and M. R. Brann.
G. Lambecht,
R. Tacke,
E. Mutschler,
Antatonist binding profiles of five cloned human muscarinic receptor subtypes. J. Pharmacol. Exp. Ther.
256: 727-733,199l. 8. Eglan, R. M., and R. L. Whiting.
Muscarinic receptor subtypes: A critique for the current classification and a proposal for a working nomenclature. J. Auton. Phurmucol. 5: 323-346, 1986.
9. Fukuda, K., T. Kubo, A. Maeda, I. Akiba, H. Bujo, J. Nakai, M. Mishina, H. Higashida, E. Nehr, A. Marty, and S. Numa.
Selective effector coupling of muscarinic acetylcholine receptor subtypes. In: Subtypes of Muscurinic Receptors IV, edited by R. R. Levine and N. J. M. Birdsall. Cambridge, UK: Elsevier, 1989, p. 411. 10. Gallacher, D. V. Control of calcium influx in cells without action potentials. News Physiol. Sci. 3: 244-249, 1988. 11. Gater, P. R., V. A. Alabaster, and I. Piper. A study of the muscarinic receptor subtype mediating mucus secretion in the cat trachea in vitro. PuZm. PhurmucoZ. 2: 87-92, 1989. 12. Gunst, S. T., J. Q. Stropp, and N. A. Flavahan. Muscarinic
J. Biol. Chem. 260: 3440-3450,1985. 14. Ishihara, H., S. Shimura, N. Ishide, M. Miura, M. Sato, H. Sasaki, and
M. Satoh, T. Masuda, T. Takishima. Intracel-
lular calcium concentration in acinar cells of feline tracheal submucosal glands. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3) L345-L350,1990. 15. Leikauf, G. D., I. F. Ueki, and J. A. Nadel. Autonomic regulation of viscoelasticity of cat tracheal gland secretions. J.
mine on cardiac and pulmonary muscarinic receptors in the rabbit. Br. J. Phurmucol. 97: 506-512, 1989. 18. Mak, J. C. W., and P. J. Barnes. Autoradiographic
visualization of muscarinic receptor subtypes in human and guinea pig lung.
Am. Rev. Respir. Dis. 19. Munson, P. J., and
141: 1559-1568, P. Rodbard.
1990.
LIGAND, a versatile computerized approach for characterization of ligand-binding systems.
Anal. Biochem. 107: 220-239,198O. 20. Sasaki, T., S. Shimura, K. Ikeda, H. Sasaki, and T. Takishima. Sodium efflux from feline tracheal submucosal gland in feline trachea. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L112-L117,1990. 21. Sasaki, T., S. Shimura, H. Sasaki, and T. Takishima. Effect
of epithelium on mucus secretion from isolated submucosal glands from feline trachea. J. Appl. Physiol. 66: 764-770, 1989. 22. Scanlon, M., D. A. Williams, and F. S. Fay. A Ca2+-insensitive form of fura- associated with polymorphonuclear leukocytes. J. Biol. Chem. 262: 6308-6312,1987. 23. Shimura, S., T. Sasaki, K. Ikeda, H. Sasaki, and T. Takishima. VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands. J. Appl. Physiol. 65: 2537-2544, 1988. 24. Shimura, S., T. Sasaki, K. Ikeda, K. Yamauchi, H. Sasaki, and T. Takishima. Direct inhibitory action of glucocorticoid to glycoconjugate secretion from airway submucosal glands. Am. Rev. Respir. Dis. 141: 1044-1049, 1990. 25. Shimura, S., T. Sasaki, H. Okayama, H. Sasaki, and T. Takishima. Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea. J. Appl. Physiol. 63: 646653,1987. 26. Shimura, S., T. Sasaki, H. Sasaki, and T. Takishima. Contractility of isolated single submucosal gland from trachea. J. Appl. Physiol. 60: 1237-1247, 1986. 27. Watson, M., W. R. Roeske, and H. I. Yamamura. [3H]piren-
zepine and (-) - [“HI quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes. J. PhurmucoZ. Exp. Ther. 236: 419-427,1986. 28. Watson, M., H. I. Yamamura, and W. R. Roeske. A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes. Life Sci. 32: 3001-3011,1983. 29. Yang, C. M., J. M. Farley, and T. M. Dwyer. Muscarinic stimulation of submucosal glands in swine trachea. J. Appl. Physiol. 64: 200-209,1988. 30. Yang, C. M., J. M.
Farley, and T. M. Dwyer. Acetylcholinestimulated chloride flux in tracheal submucosal gland cells. J. Appl. Physiol.
65: 1891-1894,
1988.
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1040-0605/92
$2.00
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gland cells of swine trachea and speculated that electrolyte and/or water secretion is stimulated via activation of M1 receptors and mucin secretion is stimulated via activation of M3 receptors. Meanwhile, Gater et al. (11) have reported that the muscarinic receptor subtype mediating mucus secretion in cat trachea has an intermediate affinity for pirenzepine, suggesting an “intermediate” between M1 and M2 receptor subtypes. We have shown that muscarinic stimulation produces a rise in intracellular calcium ion concentration ( [ Ca’+]J, resulting in mucus glycoprotein secretion (14) and electrolyte secretion (20). However, to the best of our knowledge, there have been no reports on the kind of muscarinic receptor subtype that mediates [Ca2+]i in airway submucosal gland cells. Therefore, we examined the effects of Ml-, M2-, and M3-receptor antagonists on [Ca2+]; rise by cholinergic stimulation using isolated feline tracheal submucosal glands, which enables us to examine submucosal gland secretion in a well-defined condition by excluding possible effects from surrounding tissues (2 1). MATERIALS
AND
METHODS
We measured methacholine (MCh)-induced [Ca”+]; rise in the presence and absence of each of four muscarinic receptor antagonists: atropine, pirenzepine (PZ), 11( [ 2- (diethylamino)methyl-l-piperidinyl]acetyl)-5,11-dihydro-6H-pyreido(2,3-b)(1,4)-b enzo-diazepin-6-one (AF-DXllG), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP methiodide) and compared this with radiolabeled glycoconjugate secretion and 22Na efflux from isolated feline tracheal submucosal glands by the methods described in our previous reports (2025). Trichloroacetic acid (TCA) -precipitable glycoconjugate secretion represents mucus glycoprotein (MGP) secretion (24)) and 22Na efflux represents electrolyte and/or fluid secretion from submucosal glands (20). Isolation of submucosal glands from feline trachea. Submucosal glands were isolated from feline tracheae by the method previously reported by us (26). The tracheae were removed from 34 cats (2.3-3.9 kg body w-t) under light anesthesia with intramuscular ketamine hydrochloride and intravenous thiopental sodium (30 mg/kg). Immediately after removal, submucosal glands were isolated under a stereoscopic microscope with two pairs of sharpened tweezers and microscissors. To avoid tissue damage during isolation, care was taken to isolate the submucosal gland by picking up some of the connective tissue attached to the glands. The entire procedure was performed in a modified Krebs-Ringer bicarbonate (KRB) solution containing the following (in mM): 125 NaCl, 5 KCl, 1.2 Mg CL, 1 CaC12, 25 NaHC03, 1.2 NaHP04, and 11 glucose with bubbling of 5% C02-95% O2 and pH 7.4 at 20°C. Measurement of 3H-labeled glycoconjugate secretion. The isolated glands were incubated with 2 ml of medium 199 containing D [6-“H] glucosamine hydrochloride (2.5 &i/ml, sp act 27-40 the American
Physiological
Society
L223
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L224
MUSCARINIC
RECEPTORS,
[CA2+]i
IN
Ci/mmol; Amersham, Tokyo) for 16 h and then allowed to incubate further at 37°C for two successive Z-h periods (period I and period II) in 2 ml of culture media without a radiolabeled precursor. Each antagonist was added to the media 15 min before the end of period I. In period II, culture was done in the presence of MCh (10v5 M) and each muscarinic antagonist. At the end of each period, the culture media were harvested and precipitated with 10% TCA and 1% phosphotungstic acid (PTA) at 4°C overnight. The precipitated radioactivity was measured in a liquid scintillation counter (LSC-1000, Aloka, Tokyo). The ratio of precipitated radioactivity [disintegrations/ min (dpm)] from period II to that from period I was determined for each sample and was termed the secretory index. The effect of antagonists was determined by comparing the secretory indexes of the antagonist-treated samples with those of matched control samples. Measurement of [Ca2”/i. [Ca”‘]; in the isolated glands was determined by the fura 2 method with a microscopic system CAM-220 (Japan Spectroscopic, Tokyo). The isolated glands were incubated at room temperature for 2-4 h in the loading solution, which contained 5 PM of the acetoxymethyl ester of fura 2 (fura 2/AM) and 0.2% of a detergent, Cremophor EL, to increase the solubility of fura 2/AM. After fura 2 loading, the glands were further incubated in fura 2-free KRB. Each gland was then put on a cover glass (0.12-0.17 mm of thickness) on the stage of an inverted microscope, which was warmed to 37°C by tungsten electrical coils. The microscopic field was adjusted to the acinar portion of the glands with a magnification of x400. The excitation wavelengths were 340 and 380 nm, and the emission wavelength was 500 nm. The fluorescence ratio was recorded as a function of time and converted to [Ca2+]; by the method of Grynkiewicz et al. (13) and Scanlon et al. (22) by the equation [Ca2+]i = (R - R,i,)/(R,,, - R) &* S where R is the experimentally determined fura 2 fluorescence ratio, and the dissociation constant (&) is assumed to be 224 nM (13). R,,, and Rmin were determined by the addition of 10s5 M ionomycin and 5 mM ethyleneglycol-bis(@-aminoethyl ether)N,N,N’,N’ tetraacetic acid (EGTA), respectively. The constant S is the ratio of fluorescence at 380 nm in a Ca2+-free solution to that in a Ca2+-containing solution in the presence of ionomycin. Measurement of 22Na efflux. Ten to twenty submucosal glands were incubated in 5% C02-95% O2 bubbled KRB containing 5 &i/m01 of 22NaC1 (sp act 502.6 mCi/mg, New England Nuclear, Boston, MA) at 37°C for 2 h as in our previous report (20). 22Na-loaded glands were transferred to a perfusion apparatus of a vertical acryl resin tube. The perfusate, a KRB solution gassed at 37°C either with or without drugs, was continuously pumped to the glands at a constant flow rate, and samples of effluent were collected at 18-s intervals over a period of 10 min. The 22Na radioactivity in collected effluent samples and that remaining in the gland samples at the end of the experiments were determined with an auto-gamma counter (ARC-500, Aloka, Tokyo). Drugs were applied to the glands by changing the perfusate to one containing a defined concentration of the drug required. The instantaneous rate constant for each individual sampling period was calculated as described previously (20). Stimulation with MCh was performed in the 22Na-loaded glands after 5 min of perfusion. The change in the instantaneous rate constant with MCh was estimated by comparing the baseline rate constant (mean of the rate constants during 3-5 min) with that of the maximal response in the presence or absence of each antagonist. Receptor binding assays. Two hundred fifty isolated glands from nine animals were used for the receptor-binding assays. Isolated glands were kept at -80°C until use. For membrane preparation, the feline tracheal submucosal glands were homogenized in ice-cold 50 mM Na-K phosphate
TRACHEAL
SUBMUCOSAL
GLANDS
buffer, pH 7.4 (40 mM K2HP04, 10 mM NaH2P04) with a Polytron homogenizer (setting 5) for three times (20 s each). The homogenate was subjected to a low-speed centrifugation at 1,200 g for 5 min. The supernatant was further centrifuged at 35,500 g for 15 min at 4°C. The final pellet was resuspended in the same buffer to a concentration of 8-10 pg protein/ml. The homogenate was used immediately for the receptor-binding assays. Protein was determined by the method of Lowry et al. W) For the radioligand binding assay, the membrane protein (810 pg) was added to the reaction mixture containing increasing concentrations of [3H] quinuclidinyl benzilate ( [ 3H] QNB, 50 Ci/ mmol; Amersham, Tokyo, Japan) in the 50 mM Na-K phosphate buffer (pH 7.4) to a total volume of 1.1 ml. Nonspecific binding was determined by the addition of I PM atropine. Samples were incubated at 25°C for 2 h, and binding was terminated by filtering the samples over Whatman GF/C glass fiber filters with the use of a Brandel cell harvester apparatus with three 5-ml washes of ice-cold buffer. The filters were placed in scintillation vials with Scintisol EX-H (Wako Pure Chemicals, Osaka, Japan), and radioactivity was counted by a Packard 2200CA scintillation counter (Packard Instruments). Competition assays were done as described above except that 1 nM [“H]QNB was added and various concentrations of a specific competitor were introduced, making a final volume of 1.1 ml. Reagents. Drugs used in this study were fura 2/AM (Dojin, Kumamoto, Japan), Cremophor EL (Nakarai Chemicals, Kyoto, Japan), ionomycin (Calbiochem, La Jolla, CA), acetyl,&methylcholine hydrochloride (methacholine), EGTA (Wako Pure Chemicals, Osaka, Japan), atropine sulfate, pirenzepine dihydrochloride (Sigma Chemical, St. Louis, MO), medium 199 (Flow Laboratory, McLean, VA), and Aquasol 2 (New England Nuclear, Boston, MA). AF-DXll6 was a generous gift from Boehringer-Ingelheim, Ingelheim, FRG, and 4-DAMP was a generous gift from Dr. R. Barlow, University of Bristol, Bristol UK. Statistical analysis. IC 5. values and Hill coefficients were determined from computerization of the logit log curve. The Cheng-Prusoff equation (5) was used to calculate inhibitor constant (Ki) values from the ICso values. & and the maximum binding capacity (B,,,) were determined by the nonlinear multipurpose curve-fitting program LIGAND (19). Data are means t SE. For multiple mean comparisons, analysis of variance and Duncan’s multiple range test were used. For mean comparison, the two-tailed paired or unpaired Student’s t test was used, and the Cochran Cox t test was used when Bartlet’s test for uniformity of variance showed it to be nonuniform. P < 0.05 was considered statistically significant. RESULTS
[Ccz’+/i rise. Ninety-five percent or more of the isolated glands responded significantly to MCh and produced significant rises in [Ca’+];. After stimulation with 10m5 M MCh, [ Ca2+]i increased from the resting level of 135 nM to 380 nM, and the increase was 344 t 36% (n = 26) of the resting level when expressed as a percentage of the peak value after stimulation to that of the resting level of each sample. After we confirmed the significant rises in [Ca2+]i in response to 10v5 M in control isolated glands, the other isolated glands from the same animals were used for the antagonist experiments. Each antagonist at 10-10-10-5 M was added to the KRB solution containing fura T&loaded isolated glands 15 min before each measurement of [Ca2+]; in response to 10V5M MCh.
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MUSCARINIC
RECEPTORS,
[CA2+]i
IN
The inhibitory effect of the antagonist was obtained by comparing the response to 10m5 M MCh in the presence of the antagonist with that to 10B5 M MCh in the absence of antagonist (control). The rise in [ Ca2+]; with MCh in the presence of each antagonist at 10-10-10-5 M was divided by that with MCh in the absence of any antagonist, and the inhibition due to antagonist was expressed as a percentage of the latter. Inhibition curves for four muscarinic antagonists on the MCh-induced [Ca”‘]i rise are summarized in Fig. 1 and Table 1. 4-DAMP produced an inhibitory curve similar to that for atropine, as shown in Fig. 1, and ICSo values for $-DAMP and atropine were 8 x IO-’ and 6 x lo-’ M, respectively. PZ and AF-DX116 yielded ICSo values of 10m7 and 6 X 10D6 M, respectively, and PZ at 10m6 M produced a significantly stronger inhibition than did AF-DX116 (P < 0.05) (Fig. 1 and Table 1). Thus 4DAMP was 100-fold more potent than PZ and l,OOO-fold [Ca’] (%)
’ rise
PO-
0
20 -
OI -;or+l
I
I -7 of antagonist
-8 Concentration
I
I
-6
-5
(log M >
Fig. 1. Inhibition curves of low5 M methacholine (MCh)-evoked intracellular calcium ion concentration ([ Ca2+]J rise for 4 muscarinic receptor antagonists: pirenzepine (o), 11{[2-(diethylamino)methyl-l-piperidinyllacetylj-5, ll-dihydro-GH-pyrido(2,3-b)(1,4)-benzo-diazepin-6one (AF-DXllG), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, w), and atropine (A). * P < 0.05, difference between 4-DAMP and pirenzepine. “f P < 0.05, difference between pirenzepine and AFDXl16. Results are means for 3-12 experiments.
TRACHEAL
SUBMUCOSAL
L225
GLANDS
more potent than AF-DX116 in antagonistic action. To understand the effects of PZ and $-DAMP on Ca2+ release from intracellular storage due to muscarinic receptor stimulation, we examined the effects of 10D7M PZ and 10B7M 4-DAMP on [Ca”‘]i rise in response to 10m5M MCh in a Ca2+-free solution. For Ca2+ depletion, 4 mM EGTA was added to the Ca2+-depleted solution, which was prepared by removing CaC12from the KRB solution. 4-DAMP (10m7M) abolished the MCh-evoked [Ca”‘]; rise (2 t 2% of control, P < 0.001, n = 4), whereas PZ (10m7M) did not significantly alter the MCh-evoked [Ca2+]i rise (122 t 40% of control, n = 5) in the Ca2+free condition. MGP secretion. The inhibitory effect of each antagonist on [3H]glycoconjugate secretion was obtained by comparing the increase in response to low5 M MCh in the presence of the antagonist with that in the absence of the antagonist, as described above. Inhibition curves for four muscarinic receptor antagonists on MCh (10S5 M) -induced MGP secretion are summarized in Fig. 2 and Table 2. $-DAMP has an inhibitory effect as potent as that of atropine, and IC50 values for 4-DAMP and atropine were 8 X lo-’ and 6 X lo-’ M, respectively. IC50 values for PZ and AF-DX116 were 10V6 and 2 X low5 M, respectively. 4-DAMP had a IOO-fold more potent inhibitory effect than did PZ and a l,OOO-fold more potent inhibitory effect than did AFDX116. Furthermore, PZ at 10B5 M produced significantly stronger inhibition than did AD-DX116 (P < 0.05, Fig. 2 and Table 2). 22Na efflux. Because the most dominant difference in antagonistic action in MCh-evoked [Ca2+]; rise or MChevoked MGP secretion between PZ and $-DAMP was observed at the concentration of low7 M of each antagonist, we examined the effects of 10m7M PZ and 10D7M 4-DAMP on MCh (low5 M)-induced 22Na efflux. Rate constants of 22Na efflux evoked by low5 M MCh were completely blocked by low7 M atropine, as shown in Fig. 3. PZ (10m7M) tended to reduce MCh-evoked 22Na-efflux from 349% of baseline rate constant (MCh alone) to 238% of baseline (MCh plus PZ), but the difference was not statistically significant. $-DAMP showed a significant inhibitory action and inhibited 22Naefflux to 130% of baseline rate constant, which was significantly lower than that with MCh alone (P < 0.01) or by PZ (P c 0.05, Fig. 3). Furthermore, 10B7 M atropine exhibited significantly lower rate constants than did 4-DAMP (P < 0.05, Fig. 3).
Table 1. Effects of each muscarinic antagonist on [Ca2+]i evoked by low5 M methacholine in isolated feline tracheal submucosal glands Antagonist
Pirenzepine AF-DXll6 4-DAMP Atropine
92.Okl9.7 95.3tl5.1 112.3kl6.0 89.2227.4
(6) (5) (5) (4)
96.2kl2.7 88.9kl7.2 81.6t25.3 82.4t17.3
(5) (5) (3) (7)
113.8tl7.1 82.8t12.7 37.ltl4.6* 20.5t8.3
Concentration,
(3) (5) (4) (8)
Data are means t SE expressed as % of controls (methacholine alone); values (diethylamino)methyl-l-piperidinyl]acetyl)-5,ll-dihydro-6H-pyrido(2,3-b)(l,4)-benzo-diazepin-6-one; * P < 0.05 compared with pirenzepine, t P < 0.05 compared peridine methiodide.
M
55.2kl7.6 88.7219.8 3.3&1.2* 8.9k2.6 in parentheses with
(12) (5) (6) (5)
15.8+2.5j76.1t19.0 9.0t5.4 1.5t0.6
(11) (6) (8) (5)
l.Ot3.6 40.7t18.0 -0.5t2.7 0.6tl.3
are numbers of experiments. AF-DXll6, 4-DAMP, 4-diphenylacetoxy-N-methylpi-
(5) (6) (4) (5) 11( [2-
AF-DXll6.
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L226 MGP (%>
MUSCARINIC
RECEPTORS,
[CA2+]i
IN
secretion I
60
1
n
0’ h--,-1
I
1
-10
-9
-8
Concentration
I
I
-7
-6
of antagonist
(log
---_A I
-5 M>
Fig. 2. Inhibition curves of 10s5 M MCh-evoked (TCA)-precipitable [“HI glycoconjugate secretion for 4 muscarinic receptor antagonists: pirenzepine, AF-DXll6,4-DAMP, and atropine. Symbols are same as in Fig. 1. * P < 0.05, ** P < 0.01, difference between 4-DAMP and pirenzepine. t P < 0.05, difference between pirenzepine and AF-DXll6. Results are means for 3-11 experiments. MGP, mucus glycoprotein.
Receptor-binding assays. [3H]QNB binding to submucosal gland homogenates was concentration and time dependent, and the specific binding was 82%. [3H]QNB saturation curves yielded a linear Scatchard plot. The & value was 67 t 7.2 pM, and B,,, was 1,500 t 62 fmol/ mg protein (n = 3). The Kd value was similar to those reported in rat cerebral cortex (42 pM from Ref. 28 and 21 pM from Ref. 27) and rat heart (21 pM from Ref. 27). These results indicate that the present receptor binding assays enable us to estimate the specific binding of [3H]QNB to the muscarinic receptors in submucosal gland cells. Ki and Hill coefficients of four different muscarinic antagonists are shown in Table 3. The Ki value of 4DAMP was similar to that of atropine and was 10m2and lOa or lessthan that of PZ and AF-DX116, respectively. DISCUSSION
The degree of inhibition by a receptor antagonist may be related to the concentration of a receptor agonist, and it is important to assessthe action of antagonists as well
TRACHEAL
SUBMUCOSAL
GLANDS
as agonists over a wide range of doses. However, our previous study (14) revealed an inhibitory action of fura 2 on mucus secretion from submucosal glands, which is dominant in the stimulation by agonists at lower concentrations. This makes it difficult to examine the shifts of the dose-response curves in [Ca2+]i of acinar cells of isolated glands and to obtain mean effective concentration (EC& of pA2 of each antagonist whereas no significant inhibitory action of fura 2 is observed when stimulated by MCh at higher concentrations (10D5 or 10W4 M). Therefore, we adopted the comparison of IC50 of each antagonist over a wide range of concentrations on [Ca2+]i rise by MCh at a relatively higher concentration of 10D5M in the present study. In this study we demonstrated that 4-DAMP, an M3receptor antagonist, is loo-fold more potent than PZ, an M1 receptor antagonist, and is l,OOO-fold more potent than AF-DXll6, an M2-receptor antagonist, in inhibiting [Ca2+]i rise by MCh in acinar cells of isolated glands. Furthermore, $-DAMP produced inhibitory curves similar to atropine, a nonspecific muscarinic receptor antagonist. These findings indicate that muscarinic agonists stimulate [Ca2+]i rise by activating Ma-muscarinic receptor subtypes in airway submucosal gland cells. Our previous report (14) indicated that MCh, especially at higher doses, produces [Ca2+]i rise both by release from intracellular storage and influx from the extracellular medium. In the present study, $-DAMP abolished a transient [Ca2+]; rise in a Ca2+-free solution, whereas PZ did not. This finding indicates that both the Ca2+ influx and its release from intracellular storage are coupled with the stimulation of MS-muscarinic receptor subtypes. It has been demonstrated that muscarinic receptor stimulation results in the breakdown of membrane phosphatidylinositol 4,5-bisphosphate and the formation of inositol 1,4,5trisphosphate (IP3) and that IP3 stimulates Ca2+ release from intracellular storage and, in the presence of inositol 1,3,4,&tetrakisphosphate (IP4), induces Ca2+ influx from the extracellular medium in other exocrine gland cells (10). The present findings provide evidence for phosphoinositide turnover in the coupling between M3-muscarinic receptor stimulation and mucus secretion in feline tracheal submucosal gland cells, similar to that in other exocrine gland cells. Our previous experiment (14) indicated that muscarinic receptor stimulation induces MGP secretion mediating the intracellular Ca2+system as a second messenger in feline tracheal submucosal glands. 4-DAMP showed the most potent inhibitory action to MCh-evoked glycoconjugate secretion among the three antagonists, an
Table 2. Effects of each muscarinic antagonist on trichloroacetic acid-precipitable [3H]glycoconjugate secretion evoked by 10B5M methacholine from isolated feline tracheal submucosal glands Antagonist lo-l0
Pirenzepine AF-DXll6 4-DAMP Atropine with
1225224.9 77.8kl9.4 81.6kl6.4 114.3kl5.5
1o-g
(5) (5) (5) (6)
102.lt24.8 8l.Otl0.8 99.lk21.5 128.2tl3.2
Data are mean % controls (methacholine pirenzepine, $ P < 0.05 compared with
1o-8
(5) (11) (5) (4)
110.3tl6.0 82.0t14.6 25.lt4.2* 38.2kl5.2
alone) f: SE; values AF-DXll6.
Concentration,
M 1o-7
(10) (4) (3) (9)
in parentheses
116.9tl9.3 86.2t17.6 11.2&9.3? 0.75kl2.1 are numbers
lo+
(10) (4) (4) (4)
1o-5
68.1t7.9 76.0t21.8 -7.7+1.3”r -14.6kl.7
of experiments.
(10) (5) (4) (3) *
16.3+7.5$ 55.9kl5.3 1.3kl.8 -14.3t4.3
(4) (11) (4) (6)
P < 0.05, “f P < 0.01 compared
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MUSCARINIC
RECEPTORS,
[CA2+]i
IN
TRACHEAL
SUBMUCOSAL
L227
GLANDS
glands. Autoradiographically, the coexistence of M1 and MS receptors over human airway submucosal glands has 400been demonstrated by Mak et al. (18). Yang et al. (30) have reported that acetylcholine caused a Ca2+-dependent decline in intracellular 36C1of submucosal gland cells 051 rP< of diisopropylfluorophosphate-treated swine trachea, 300and this loss of intracellular Cl was completely blocked by PZ or atropine. From these observations, they speculated that electrolyte (or fluid) secretion from airway 200 submucosal glands is coupled mainly with M1 receptor stimulation. In contrast, our present study shows the main role of M3 receptors in electrolyte secretion from feline tracheal submucosal glands. The difference belootween the results of Yang et al. (30) and our own may be due to differences in the species used for experiment. Another possible explanation is that Yang et al. used a single and relatively higher concentration of PZ in their OMCh MCh MCh MCh experiment and also did not examine the effects of M3 alone t t t receptor antagonist. Although PZ is considered to be a PZ 4 DAMP ATR selective antagonist for M1 receptors, the selectivity is Fig. 3. MCh (lo-“)-evoked 22Na efflux from isolated glands in absence lost at higher doses and it acts as an antagonist for MB or presence of each antagonist [10m7 M pirenzepine (PZ); 10v7 M 4receptors in the heart and for M3 receptors in smooth DAMP, or 10s7 M atropine (Atr)]. Results are expressed as % of muscle (8, 13, 17). For example, Gunst et al. (12) have baseline rate constant and means & SE for 3-5 experiments. ** P < reported that 10m6M PZ antagonizes M3 receptors of 0.05, compared with MCh alone. canine tracheal smooth muscle, whereas 10m8M PZ does action similar to the [Ca2+]i rise by MCh, in the present not. Our present experiment also demonstrates signifistudy. TCA-precipitable glycoconjugates secreted from cant inhibition by PZ at 10V5 and/or 10D6 M of both isolated glands represent MGP (24). Therefore, the pres- [Ca2+]i rise and MGP secretion, compared with that by ent finding indicates that MGP secretion evoked by AF-DXll6, as shown in Figs. 1 and 2. Both Mak et al. muscarinic stimulation is mediated by an MS-subtype (18) and Yang et al. (30) adopted a single concentration muscarinic receptor. of a relatively higher dose (low6 M) for their experiments. Previous reports, including our own (6, 15, ZO), have M3 receptors in airway glandular cells may be more shown that submucosal glands secrete not only MGP sensitive to PZ than those in smooth muscles. (mucin) but also electrolyte with fluid. Furthermore, the The present experiments including receptor-binding latter is detected by determining 22Na efflux from iso- assays indicate few or no M2 receptors. For example, the lated submucosal glands of feline trachea, which repre- present receptor-binding assays showed that the Ki value sents Na-K pump activity of the secretory cells (20). of AF-DXll6 was 660 nM (Table 3), which is much Although a limited experiment, 4-DAMP was the most larger than Ki values reported in the tissues possessing potent in inhibitory action on 22Na efflux from isolated M2 receptors. Watson et al. (27) reported that the K; feline submucosal glands. This indicates that electrolyte value of AF-DXll6 in rat heart is 39 nM, determined by (or fluid) secretion is also mediated mainly by MS-mus- [3H]QNB binding assay. After the submission of this carinic receptor subtypes in the submucosal gland. Meanmanuscript, Dorge et al. (7) reported that 4-DAMP has while, the inhibition by PZ was smaller than that by the an antagonistic action to M1 receptors in a degree similar control (MCh alone), and inhibition by 4-DAMP was to the action to M3 receptors. Our receptor binding assay significantly smaller than that of Atr. As well, PZ seems showed that the Hill coefficient value of PZ was 0.71, to have a stronger inhibitory action on [Ca2+]i rise than suggesting two or more populations of the binding recepthat on MGP secretion by MCh, as seen in Figs. 1 and tors. It is also possible that M1 and M3 receptors coexist 2. These findings do not eliminate the possibility that an in submucosal gland cells as indicated by previous reports M1 receptor has a small role in the electrolyte secretion (11, 18, 29). However, ID50 values for PZ in glycoconjufrom airway submucosal glands and that it may be too gate secretion and [Ca2+]i rise are 10D6 and 10m7M, small to detect by the present experimental design. respectively. These values are much larger than 1.8 X Some investigators have suggested the presence of M1 IO-’ M, which is reported as an apparent & value of M1 receptor in addition to M3 receptor in airway submucosal receptors for PZ (9). Therefore, M3 receptors functionally **Na-efflux (Oh of
control
)
Table 3. Dissociation constants and Hill coefficients of different muscarinic antagonists for muscarinic receptor subtypes Antagonist
Dissociation constants, Hill coefficients, nH
nM
Dissociation constants were derived from are means t SE of 3 experiments performed
Pirenzepine
AF-DX116
22t2.6 0.71~0.003
660t92 1.03kO.12
4DAMP
Atropine
0.62kO.089 0.65t0.05
0.29t0.044 0.96t0.07
the experimental values for half-maximal inhibitory concentration in duplicate. For each curve, 6-9 concentrations were used.
as described
in METHODS.
Values
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L228
MUSCARINIC
RECEPTORS,
[CA2+]i
IN TRACHEAL
SUBMUCOSAL
GLANDS
play a primary role in mucus secretion and [ Ca2+]i rise in feline tracheal submucosal glands. In conclusion, in the present study, we demonstrated that muscarinic stimulation induces MB-receptor-operated [Ca”‘]i rise and MGP and electrolyte (or fluid) secretion from the secreting cells of feline tracheal submucosal glands. These findings are important in the development of effective therapeutic methods for mucus hypersecretion in various pulmonary diseases.
receptor reserve and P-adrenergic sensitivity in tracheal smooth muscle. J. Appl. Physiol. 67: 1294-1298, 1989. 13. Grynkiewicz, G., M. Poenie, and R. Y. Tien. A new generation of Ca2+ indicators with greatly improved fluorescence properties.
We gratefully acknowledge Professor Akinori Nishiyama and Dr. Yoshitaka Saito at the Department of Physiology, Tohoku University School of Medicine, for their helpful advice and discussions, Dr. Ronald Scott and Elizabeth Crittenden for correcting the English, and Kumiko Shibuya for typing the manuscript. This study was supported by Scientific Grant 01570422 from the Ministry of Education, Science, and Culture of Japan. Address for reprint requests: T. Takishima, First Department of Internal Medicine, Tohoku University School of Medicine, l-l Seiryomachi, Aoba-ku, Sendai 980, Japan.
Appl. Physiol. 56: 426-430, 1984. 16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193: 265-275,195l. 17. Maclagen, J., and D. Faulker. Effect of pirenzepine and galla-
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