Center for Oral Biology and the Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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Rat sublingual gland
M1 and M3 muscarinic receptors each directly
activate exocrine secretion. To investigate the functional role of
coreceptor expression, we determined receptor-G protein coupling.
Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by
pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both
carbachol-stimulated high-affinity GTPase activity and the GTP-induced
shift in agonist binding are insensitive to CTX or PTX. Carbachol
enhances photoaffinity labeling
([-32P]GTP-azidoaniline) of only 42-kDa proteins that
are subsequently tractable to immunoprecipitation by antibodies
specific for G
q or G
11 but not
G
12 or G
13. Carbachol-stimulated
photoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate
(PIP2) hydrolysis is reduced 55% and 60%, respectively,
by M1 receptor blockade with m1-toxin.
G
q/11-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar
ratios of receptors to G
q and G
11.
Although simultaneous activation of M1 and M3
receptors is required for a maximal response, both receptor subtypes
are coupled to G
q and G
11 to stimulate
exocrine secretion via redundant mechanisms.
salivary glands; muscarinic cholinergic receptors; mucous cells; m1-toxin; Gq/11; exocrine secretion
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INTRODUCTION |
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SALIVARY GLANDS of the oral cavity are numerous and diverse and are composed of either one or both of two principal acinar exocrine cell types, serous and mucous cells. In rodents and humans, mucous exocrine cells predominate within acinar structures and receive robust parasympathetic innervation with a paucity of sympathetic innervation. Correspondingly, unlike serous glands, both fluid secretion (20) and exocrine secretion of mucin glycoproteins by mucous glands (5) are primarily under muscarinic cholinergic control. Previously, we identified equivalent amounts of M1 and M3 muscarinic receptor subtypes in membranes from either whole rat sublingual glands or isolated acini (27). Furthermore, both muscarinic receptor subtypes appear to be associated directly with mucous acinar cells and are required for maximal exocrine secretion (5). The coexpression and regulatory function of muscarinic receptors within mucous acini may or may not extend to signaling events at the postreceptor level. For example, M1 and M3 receptors may each couple potentially to different guanine nucleotide binding proteins (G proteins), even when expressed in the same cell (8).
At least 19 separate G protein -subunits have been identified in
mammalian tissues and are segregated into four families on the basis of
primary sequence homology: G
s, G
i,
G
q, and G
12 (11).
Cholera toxin (CTX) catalyzes the ADP ribosylation of the
G
s family and G
t(1 and 2), resulting in
the loss of intrinsic GTPase activity (14). Pertussis
toxin (PTX) ADP ribosylates a cysteine residue near the carboxyl
terminus of G
i, G
o, G
t, and G
gus, preventing subsequent G protein and receptor
interactions (14). In studies of cell lines expressing
M1 or M3 receptors, both receptor subtypes are
linked predominantly to G
q/11 to activate phospholipase
C (PLC) (8, 14). In addition, both receptor subtypes may
activate PTX-sensitive G proteins (21) and stimulate PLC
through G
dimers (16). In rat parotid glands,
M3 receptors couple to both G
q and
G
i1 (6). Thus, in sublingual glands, coupling of either M1 or M3 receptors to other
G proteins in sublingual acini cannot be ruled out, especially given
the predominance of muscarinic receptors in regulating mucous acinar
cell functions.
To provide insights into the functional role of receptor coexpression
in sublingual glands, we therefore investigated the coupling of
M1 and M3 receptors in sublingual membranes to
G protein -subunits. The coupling and toxin sensitivity (PTX and
CTX) of receptors to G proteins was assessed by high-affinity
GTPase activity and the GTP-induced shift in carbachol
high-affinity binding sites. Photoaffinity labeling by
[32P]GTP-azidoaniline and antisera specific for
-subunits was used to detect candidate subunits coupled to
muscarinic receptors and to identify
-subunits linked to
carbachol-induced phosphoinositide hydrolysis. The contribution of
M1 receptors to carbachol-induced responses was determined
by using the pseudoirreversible and highly specific M1
receptor antagonist m1-toxin, a 64-amino acid peptide isolated from the
venom of the Eastern green mamba, Dendroaspis angusticeps
(23).
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MATERIALS AND METHODS |
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Materials.
Specific pathogen and sialodacryoadenitis virus-free male Wistar rats
(2 mo old, 150-175 g) were obtained from Charles River Laboratories (Kingston facility, Stone Ridge, New York). PTX and CTX
were from List Biological Laboratories. Carbachol, atropine, ATP, GDP,
NAD, phosphocreatine, creatine phosphokinase,
5'-adenylyl-,
-imidodiphosphate (AppNHp), thymidine, benzamidine,
EDTA, EGTA, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl
(EDAC-HCl), triethylammonium bicarbonate, 2-(N-morpholino) ethanesulfonic acid (MES), HEPES, 14C-methylated protein
markers, and phosphatidylinositol 4,5-bisphosphate (PIP2) were from Sigma Chemical. Dithiothreitol (DTT)
was obtained from United States Biochemical, and GTP, guanosine
5'-O-(3-thiotriphosphate) (GTP
S), aprotinin, leupeptin,
and phenylmethylsulfonyl fluoride (PMSF) were from Boehringer Mannheim
Biochem. Protein A-agarose was obtained from Calbiochem. Immobilon-PVDF
(polyvinylidene difluoride) was obtained from Millipore. Cappel Rabbit
IgG was obtained from ICN Pharmaceuticals, polyethylenimine-cellulose
plates were from J. T. Baker, and 4-azidoaniline-HCl was from
Fluka. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) were
from Avanti Polar Lipids; [125I]-labeled rProtein A (81.1 µCi/µg), [3H]PIP2 (6.0 Ci/mmol),
[
-32P]GTP (3,000 Ci/mmol), [
-32P]GTP
(6,000 Ci/mmol), and [32P]NAD (800 Ci/mmol) were from
Dupont NEN; and [3H]-N-methyl-scopolamine
(NMS) was from Amersham International. Recombinant proteins, rat
G
q, and human G
11 were obtained from Chemicon International. Rabbit antibodies (IgG fractions, 200 µg
IgG/ml) D17, E17, S20, and A20, as well as their blocking proteins, were obtained from Santa Cruz Biotechnology. E17 was raised to amino
acids 13-29 of the amino-terminal domain unique to
G
q. D17 was raised to amino acids 13-29 of the
amino-terminal domain unique to G
11. A20 and S20 were
raised to amino acids 2-21 of the amino-terminal domain of
G
13 and G
12, respectively. Rabbit antisera W082, B825, and Z811 were generous gifts from Dr. Paul Sternweis (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX). W082 was raised to a synthetic peptide representing the internal amino acid sequence 115-133, unique to
G
q (22). Z811 was raised to a synthetic
peptide of the common carboxy-terminal sequence 345-359 for
G
q and G
11 (22). B825 was
raised to a peptide spanning the internal sequence 114-133 of
G
11 (13). Antibody CT92, kindly provided by
Dr. Dianqing Wu (Department of Pharmacology, University of Rochester
Medical Center, Rochester, NY), was raised against a synthetic peptide to the internal sequence 116-132 of G
14
(1). Antisera AS233 and AS343 (25) were
raised to synthetic peptides sharing distinct carboxy-terminal
sequences unique to G
12 (370) and
G
13 (367), respectively, and were
kind gifts from Dr. Karsten Spicher (Institut für Pharmakologie,
Freie Universität Berlin, Thielallee, Berlin, Germany).
Membrane preparation.
Rats were killed by exsanguination after CO2
anesthesia. Sublingual glands were removed quickly and kept in
ice-cold L-15 medium until all glands were obtained. The glands were
dissected at 4°C to remove remaining fat, connective tissue, and the
hilum region and were then rinsed briefly in 20 mM Tris · HCl,
pH 7.5, 1 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 0.2 mM PMSF (Tris-EDTA buffer), minced, and homogenized in 10 volumes of
Tris-EDTA buffer with a glass Dounce homogenizer. The homogenates were
filtered through 250-µm nylon mesh (Small Parts) and spun at 25,000 g for 25 min. The resulting pellets were further homogenized
(6 × 30 s) with a BioHomogenizer (Biospec Products) at a
high setting, pelleted as before, and washed with and resuspended in
Tris-EDTA buffer. Protein content was determined by the method of
Bradford, as described previously (5), with the use of
bovine serum albumin (BSA) as standard. Aliquots (2 mg/ml) were stored
at 80°C until use.
ADP ribosylation. PTX and CTX were preactivated for 30 min at 30°C with 20 and 50 mM DTT, respectively, dissolved in 20 mM Tris-EDTA buffer (pH 7.5). Activated toxin and [32P]NAD (5 µCi/assay) were mixed with ADP-ribosylation buffer consisting of 1 mM EDTA, 2.5 mM MgCl2, 100 mM NaCl (for PTX only), 1 mM ATP, 10 µM GTP, 2.5 mM DTT, 10 mM thymidine, 10 µM NAD, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 0.2 mM PMSF, and 20 mM Tris · HCl, pH 7.5, and the reaction was initiated by adding the membrane suspension (3 mg/ml resuspended in ADP-ribosylation buffer). The reaction was terminated after 60 min with ice-cold Tris-EDTA buffer, followed by spinning (12,000 g for 10 min, 4°C). Membrane pellets were subjected to SDS-PAGE and autoradiography. In competition, photoaffinity labeling and GTPase assays for bacterial toxin pretreatment of membranes were conducted with and without [32P]NAD at final concentrations of 25 µg/ml PTX or 10 µg/ml CTX, and the ratios of toxins to membrane proteins were kept constant at 10 µg PTX/mg protein and 4 µg CTX/mg protein.
GTPase assay.
Sublingual membranes were washed twice by centrifugation (12,000 g for 10 min, 4°C) with 30 volumes of ice-cold assay
buffer (50 mM triethanolamine-HCl, pH 7.4, 0.2 mM EGTA, 10 µg/ml
leupeptin, 5 µg/ml aprotinin, and 0.2 mM PMSF). To assay GTPase
activity, we used the method described by Ghodsi-Hovsepian et al.
(10). Membranes (5 µg) were incubated at 25°C for 5 min with or without muscarinic agonist or antagonist in assay buffer
supplemented with 100 mM NaCl, 5 mM MgCl2, 0.2% BSA, 5 mM
phosphocreatine, 1 mM App(NH)p, 50 U/ml creatine phosphokinase, 0.25 mM
ATP, and 1 mM DTT. The reaction was started by addition of 2 µl of
12.5 µM GTP containing 0.5 µCi [-32P]GTP (final
volume 50 µl) and stopped with 1 ml of 5% (wt/vol) Norit A suspended
in ice-cold 50 mM NaH2PO4 (pH 4.5). Tubes were centrifuged (12,000 g for 10 min, 4°C), and a 60-µl
aliquot of each supernatant was counted in 10 ml of scintillation
fluid. Results are presented as high-affinity GTPase activity, which is
defined as the difference between total and nonspecific hydrolysis of
[
-32P]GTP. Nonspecific GTPase activity was determined
in the presence of 50 µM unlabeled GTP. In control experiments, total
GTPase activity in the presence of 1 mM carbachol was linear for at
least 10 min (not shown). As a result, all subsequent assays were
conducted for 10 min. Spontaneous release of
32Pi (in the absence of membranes) accounted
for <1.5% of total added radioactivity.
Synthesis and purification of
[-32P]GTP-azidoaniline.
[
-32P]GTP-azidoaniline was synthesized and purified as
described by Fields et al. (9). Briefly,
[
-32P]GTP was evaporated under a mild nitrogen stream
and dissolved with 3.6% EDAC-HCl in 0.1 M MES (pH 5.6).
Nonradiolabeled GTP was added, and the mixture was allowed to set for
10 min. We then added 4% 4-azidoaniline-HCl suspended in peroxide-free
1,4-dioxane and incubated the solution for 16 h at room
temperature in the dark with constant rotation. The reaction was
terminated by extraction of unreacted azidoaniline three times with 200 µl of water-saturated ethyl acetate. The water phase was spotted onto
a flexible polyethylenimine-cellulose plate (5 × 20 cm), and
thin-layer chromatography (TLC) was performed at room temperature in
the dark by using 0.8 M triethylammonium bicarbonate buffer (pH 7.5) as
the mobile phase. TLC-purified [
-32P]GTP-azidoaniline
was detected by autoradiography, eluted with 0.5 M NaCl, lyophilized,
and reconstituted. Radioactivity was determined, and aliquots were
stored at
70°C. Similar procedures were followed for synthesis and
purification of nonradiolabeled GTP-azidoaniline, with the exception
that the TLC-separated GTP analog was detected with ultraviolet (UV)
light (360 nm).
Photoaffinity labeling.
Photoaffinity labeling was performed as described by Fields et al. (9) with minor modifications. Sublingual membranes were resuspended to 1 mg/ml in photolabeling buffer (20 mM Tris · HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2, 100 mM NaCl, and 1 mM benzamidine) and incubated for 5 min at 30°C with 100 µM of GDP plus an additional 5 min in the presence or absence of agonist or antagonist, as indicated. In some cases, membranes (1 mg/ml photolabeling buffer) were preincubated for 30 min at room temperature with 100-fold molar excess of m1-toxin (relative to membrane high-affinity pirenzepine sites). We then added 2 µl of 750 µM GTP containing 0.2-0.6 µCi of [SDS-PAGE and autoradiography.
Electrophoresis was as described previously (4) by using
4% stacking and 10% running gels in a Hoeffer minigel system. In some
cases, a lower concentration of bisacrylamide (0.12%) was used to
obtain better resolution of the isoforms of -subunits. Gels were
stained with Coomassie blue and/or silver as described (4), dried at 60°C with a gel dryer, and exposed to
Kodak XAR-5, Biomax MS, or MR film at
70°C. Autoradiograms were
scanned with either a laser densitometer (LKB 2202 UltroScan XL) or a
Digital Imaging System (IS-1000; Alpha Innotech).
Western blotting. Membrane proteins (50 µg) were subjected to SDS-PAGE, and proteins were transferred to Immobilon-PVDF membranes at 200 mA overnight with a Hoeffer mini transfer apparatus (TE 22). Under these conditions, no proteins of <70 kDa remained in the gel, as assessed by extensive silver staining. Membrane strips were rinsed twice and incubated in blotting solution (0.2% Triton X-100 and 5% nonfat dry milk in PBS) at room temperature for 1 h before overnight incubation with individual antisera diluted in blotting solution. After three rinses with blotting solution, strips were incubated 2-3 h with 500,000 cpm/ml [125I]-Protein A, rinsed, and subjected to autoradiography with Kodak Biomax MS or MR film.
Immunoprecipitation of photolabeled G proteins. Sublingual membranes were photolabeled as described in Photoaffinity labeling, and two aliquots were subjected to SDS-PAGE and autoradiography as controls in each experiment to verify carbachol-enhanced photolabeling of 42-kDa proteins. The resultant membrane pellets (90 µg per condition) were solubilized by first being incubated in 15 µl of 2% SDS for 10 min at room temperature. We then added 48 µl of ice-cold solubilization buffer (10 mM Tris · HCl, pH 7.4, 1% wt/vol Nonidet P-40, 1% wt/vol deoxycholate, 0.2% SDS, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mM PMSF, 1 µM GDP, and 10 µg/ml aprotinin), and the mixture was repipetted. Membrane solubilization appeared quantitative, because no specific G proteins were detectable in the subsequent pellets when assessed in preliminary experiments by Western analysis for each antibody used. After centrifugation (14,000 g for 10 min), the supernatant was precleared by adding 3.3 µl of 5 mg/ml purified rabbit IgG in PBS and incubating for 4 h at 4°C, followed by addition of 20 µl of Protein A-agarose (50% suspension, preequilibrated and washed with solubilization buffer + 0.5% SDS), and the mixture was incubated for 1 h at 4°C with continuous rocking. After centrifugation (14,000 g for 10 min), we added 20 µg of antibody IgG (with or without blocking peptide) and 1 ml of cold solubilization buffer to the supernatant and incubated overnight at 4°C with continuous rocking. Protein A-agarose (10 µl of a 50% suspension) was added, and incubation continued for 4-6 h. The mixture was centrifuged (14,000 g for 10 min), and the pellet was washed with 1 ml of solubilization buffer plus 0.5% SDS and then subjected to SDS-PAGE and subsequent autoradiography with Kodak Biomax MS film. In all cases, blocking antigen peptides were used at a 10:1 molar ratio with respect to antibody IgG in the solution. In controls consisting of nonimmune rabbit IgG in place of antibody, we were unable to detect radiolabeled proteins in the final immunoprecipitate, and no IgG was detected in the supernatant when subjected to SDS-PAGE and extensive silver staining.
PIP2 hydrolysis assay.
Measurement of PLC activity was as described by Gutowski et al.
(12) but with minor modifications. Membranes were diluted to 0.5 mg/ml with buffer A (1 mM EDTA, 3 mM EGTA, 100 mM
NaCl, 5 mM MgCl2, 3 mM DTT, 10 µM GDP, and 50 mM HEPES,
pH 7.2), and 10 µl of membrane suspension (5 µg protein) were added
to 40 µl of buffer B (3 mM EGTA, 80 mM KCl, 1 mM DTT, 1 µM GDP, and 50 mM HEPES, pH 7.2) containing phospholipid vesicles.
The final vesicle suspension was prepared by sonication and contained
1.5 mM PE, 1.5 mM PS, 0.15 mM PIP2, and 7,000-10,000
dpm [3H]PIP2. In some cases, buffer
B also contained GTPS, carbachol, and/or atropine. Ten
microliters of 9 mM CaCl2 were then added, and the reaction
was started by transferring tubes to a 30°C water bath. Reactions
were terminated after 12 min by placing the tubes in ice water,
followed by rapid addition of ice-cold 1% BSA (100 µl) and 10%
trichloroacetic acid (200 µl). After 5 min, the precipitates were
pelleted (12,000 g for 5 min), and 300 µl of the
supernatants were removed for determination of radioactivity. In
parallel assays, membranes were preincubated with m1-toxin or antibody
Z811 against G
q/11. In the case of antibody Z811,
membrane aliquots (0.5 mg/ml buffer A) were preincubated for
20 min at room temperature, followed by 30 min on ice in the absence
(control) or presence of either Z811 (1 µM IgG), Z811 premixed with
10 µM of the antigen peptide, or 1 µM rabbit IgG. Portions of each
membrane aliquot were then assayed in the absence or presence of
GTP
S (1 µM) with and without carbachol (1 mM). In the case of
m1-toxin, two equal membrane aliquots (0.5 mg/ml buffer A)
were preincubated for 30 min at room temperature with or without
m1-toxin (100-fold molar excess of m1-toxin relative to membrane
high-affinity pirenzepine sites) and then assayed in the presence of
GTP
S (1 µM), carbachol (1 mM) plus GTP
S (1 µM), or atropine
(100 µM) plus carbachol (1 mM) plus GTP
S (1 µM).
Radioligand binding. Membranes (20-50 µg) were resuspended in 0.5-1 ml of binding buffer (20 mM Tris · HCl, pH 7.5, 1 mM EDTA, 3 mM MgCl2, and 0.1 mM PMSF) and incubated with [3H]NMS at 4°C for 3 h to achieve steady-state binding. Incubations were stopped by addition of 4 ml of ice-cold binding buffer, followed by vacuum filtration through Whatman GF/C filters. Atropine (10 µM) was included in the incubation for determination of nonspecific binding. In competition assays, nonradiolabeled agonist (with or without guanine nucleotide) was added simultaneously with 0.5 nM [3H]NMS. Results were analyzed by using the computer programs EBDA and LIGAND, as described previously (27). The dissociation constant (Kd) for [3H]NMS in membrane preparations ranged from 0.7 to 1.1 nM, and the proportion of high-affinity pirenzepine sites ranged from 42 to 48% of total binding sites, similar to results reported previously (27).
Preparation of m1-toxin. Toxin was isolated from lyophilized venom of the Eastern green mamba, D. angusticeps (Sigma Chemical), according to the protocol described by Potter et al. (23), except that material from the initial Sephadex G-50 column was further fractionated by gel filtration chromatography on a column (1.5 × 170 cm) of Sephadex G-25 Superfine eluted with 0.1 M ammonium acetate, pH 6.8, at 4°C. This additional step serves to enrich the preparation for m1-toxin and removes a component possessing anti-rat M3 receptor activity before reverse-phase HPLC. Preparations of m1-toxin were pure as determined by SDS-PAGE and subsequent reverse-phase HPLC. One unit of m1-toxin was similar to that defined by Potter et al. (23): the minimum amount required to block 95% of the specific binding of 0.1 nM [3H]NMS to 0.2 pmol M1 receptors [rat M1-Chinese hamster ovary (CHO) cell membranes] in 10 ml of 50 mM NaH2PO4 plus 1 mM EDTA, pH 7.4, at 25°C for 45 min. Under these standard assay conditions, 1 unit of m1-toxin is equivalent to ~30 ng toxin protein and corresponds to a molar ratio of m1-toxin to receptor of ~20:1 (23). Preparations of m1-toxin were specific for the inhibition of M1 receptors as assessed under the standard radioligand binding assay conditions described above, which include 1 unit of m1-toxin and membranes from CHO cell lines expressing either human M1-M5 receptor subtypes (kindly provided by Dr. Mark R. Brann) or rat M1 or M3 receptors. In addition, 10 units of m1-toxin had no inhibitory effects on [3H]NMS binding to rat M3 receptors (200:1 molar ratio of m1-toxin to receptors).
Statistics. Unless indicated, variability is expressed as means ± SE. Statistical comparisons of results was by the Student's t-test with P < 0.05 being significant.
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RESULTS |
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To initially evaluate the effective coupling between muscarinic
receptors and G proteins in sublingual membrane preparations, we
studied the shift induced by GTPS in the affinity of the agonist carbachol for [3H]NMS binding sites. As shown in a
representative experiment in Fig. 1,
GTP
S induced a rightward shift in the carbachol competition curve.
Binding data from five separate experiments were fit to models of one,
two, and three binding sites, and the best fit was chosen when
P < 0.05 by the F test. In the absence of
GTP
S, the best fits were to a two-site model with inhibition
constant (Ki) values (means ± SE) of
5.4 ± 1.1 and 99.8 ± 17.0 µM, representing 54 ± 3 and 46 ± 3% of the total binding sites, respectively. In the
presence of 100 µM GTP
S, the best fits were to a one-site model
with a mean Ki of 129 ± 14 µM.
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A series of experiments were then conducted to determine whether
muscarinic receptors in sublingual membranes are coupled to G proteins
sensitive to PTX or CTX. We first determined the ADP ribosylation of
membrane proteins as a function of toxin concentration. CTX induced the
concentration-dependent ADP ribosylation primarily of 44-kDa proteins
within sublingual membranes as resolved by SDS-PAGE and subsequent
autoradiography. Maximal radiolabeling was achieved at 10 µg/ml CTX
(Fig. 2B). Similar experiments
were conducted with PTX, and, as shown in Fig.
3B, proteins of ~40 kDa were
radiolabeled in a concentration-dependent manner with maximal
radiolabeling at 25 µg/ml PTX. If proteins were allowed to migrate
further into the gel, PTX-dependent proteins began to resolve into two
bands of ~40 and 41 kDa, presumably representing at least two
distinct -subunits (Fig. 3C, lane 2). To
verify that PTX at 25 µg/ml and CTX at 10 µg/ml were saturating for
ribosylation of sublingual membrane proteins, we treated membrane
aliquots with and without toxin in the absence of
[32P]NAD and then reexposed them to toxin in the presence
of [32P]NAD. As a control, membrane aliquots were treated
with or without toxin in the presence of [32P]NAD.
Samples were subjected to SDS-PAGE and autoradiography to first verify
toxin-enhanced radiolabeling of membrane proteins of appropriate mass
and to determine residual radiolabeling of proteins in membranes
pretreated with toxin. In two separate experiments, autoradiographs
(1-day exposure) of control samples demonstrated radiolabeling similar
to results shown in Figs. 2 and 3, whereas radiolabeled proteins were
barely detectable by autoradiography after a 10-day exposure of samples
from membranes pretreated with each toxin (not shown). On the basis of
these combined results, we used PTX and CTX at 25 and 10 µg/ml,
respectively, in further experiments to distinguish whether
toxin-sensitive G proteins play a role in carbachol-stimulated
signaling events in sublingual membranes.
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PTX selectively ADP ribosylates -subunits of target G protein
holotrimers rather than free
-subunits (29). Therefore, to test coupling of sublingual muscarinic receptors to PTX-dependent G
proteins, we incubated sublingual membranes before ADP ribosylation with or without agonist (1 mM carbachol) for 15 min at room temperature in ADP-ribosylation buffer that contained 10 µM GTP. Agonist was maintained in the buffer during subsequent ADP ribosylation by PTX in
the presence of [32P] NAD. Under these
conditions, a proportion of those G proteins that couple to muscarinic
receptors would be expected to be activated. Accordingly, if muscarinic
receptors are indeed coupled to PTX-sensitive G proteins, then the pool
of receptor-coupled
-subunits in the heterotrimeric state, and hence
available to PTX-catalyzed ADP ribosylation, should be less than in the
absence of carbachol. In contrast, carbachol had no apparent effect on
PTX-dependent radiolabeling of either 40- or 41-kDa proteins (Fig.
3C, compare lanes 2 and 3). As a
control, PTX-catalyzed ADP ribosylation was completely inhibited when
membranes were incubated in the presence of 100 µM GTP
S (Fig.
3C, lane 4). In additional competition binding experiments, we found that PTX-catalyzed ADP ribosylation of sublingual membrane proteins also had no detectable effect on the shift in carbachol binding affinity induced by GTP
S (see Fig.
4).
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As another means to distinguish coupling of sublingual muscarinic
receptors to G proteins sensitive to PTX or CTX, we determined whether
either toxin attenuated agonist-enhanced high-affinity GTPase activity.
In an initial experiment, GTPase activity was induced by carbachol in a
concentration-dependent manner, the effect of which was maximal at 1 mM
with an apparent EC50 of ~7 µM (Fig.
5A). We then assayed
high-affinity GTPase activity in response to 1 mM carbachol after prior
ADP ribosylation of membranes with maximal concentrations of either PTX
or CTX. Basal GTPase activity was reduced after treatment of membranes
with each toxin (Fig. 5B), suggesting that G proteins
susceptible to either PTX or CTX contribute to the total basal
high-affinity GTPase activity. In contrast, the net increase in
carbachol-mediated GTPase activity was unaffected by pretreatment
of membranes with either toxin (Fig. 5B).
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Agonist-enhanced photoaffinity labeling of membrane proteins with
[32P]GTP-azidoaniline is a valuable tool in identifying
the coupling of receptors to G proteins (9). In initial
experiments with rat sublingual gland membranes, we found that multiple
bands were radiolabeled with [32P]GTP-azidoaniline in the
absence of agonist, including bands of 74, 51, 46, 36, and 23 kDa and a
predominant band at 42 kDa (Fig.
6A, lane 1). In the
presence of carbachol, we consistently (10 separate experiments)
observed agonist-enhanced photoaffinity labeling of only 42-kDa
proteins (Fig. 6A, lanes 2 and 3).
Photoaffinity labeling of all proteins was unaffected by 100 µM ATP
but inhibited completely by either 30 µM guanosine
5'-O-(2-thiodiphosphate) (not shown) or 100 µM GTPS
(Fig. 6A, lanes 4 and 5). Furthermore, carbachol-enhanced photoaffinity labeling was also insensitive to
pretreatment of sublingual membranes with either 25 µg/ml PTX or 10 µg/ml CTX (not shown). Enhancement of photoaffinity labeling of the
42-kDa band in the presence of carbachol was concentration dependent,
readily detectable at 0.1 µM, and maximal at ~100 µM carbachol
(Fig. 6B). The average maximal intensity of radiolabeling induced by carbachol was nearly 60% above basal levels as assessed by
densitometric scanning of autoradiographs (Fig. 6B).
In two separate experiments, we found that photoaffinity labeling
enhanced by 1 mM carbachol was completely blocked by concentrations of atropine of 0.1 µM or higher (not shown). To assess the contribution of muscarinic M1 receptors to carbachol-enhanced
photoaffinity labeling, we first treated sublingual membranes with a
200-fold molar excess of m1-toxin. Basal levels of photoaffinity
labeling was unaffected by m1-toxin, whereas carbachol-enhanced
labeling was reduced 55% (Fig. 7).
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At this stage, our combined results indicated that both M1
and M3 receptors couple to G proteins with -subunits of
~42 kDa and are insensitive to both PTX and CTX. We therefore
conducted Western blot analysis to identify candidate
-subunits
within sublingual membranes that may couple to muscarinic receptors. Possible candidates of known G protein
-subunits were considered that satisfied the following criteria: 1) insensitive to
both PTX and CTX; 2) ~42 kDa in electrophoretic mobility;
and 3) previously demonstrated to be expressed in epithelial
tissues. Candidates include a member (or members) of the Gq
family, the G
12 family, and the PTX-insensitive
Gi family member G
z. Previous studies demonstrated that G
16 and its mouse homologue,
G
15, are restricted to hematopoietic tissues
(28), whereas G
z is distributed primarily to platelets and neurons (17). Thus we did not probe for
G
16 or G
z. We did probe with antibodies
selective for G
q, G
11, G
14, G
12, and G
13. Results
are shown in Fig. 8. Antibody Z811, raised against the carboxy-terminal region common to both
G
q and G
11 (22), reacted
strongly to proteins of ~42 kDa in sublingual membranes (Fig.
8A, lane 1). Similar results (Fig. 8A,
lane 3) were obtained with the G
q-specific
antisera W082 (22) and E17 (not shown). To probe for
G
11, we used antibody D17, raised to a peptide
equivalent to amino acids 13-29 of the amino-terminal domain
unique to G
11. As shown in Fig.
8A, lane 5, this antibody reacted with 42-kDa
proteins as well as several lower and higher mass proteins in
sublingual membranes. Reactivity was blocked by preabsorption with the
antigen peptide (Fig. 8A, lane 6). Similar
results were obtained with membranes from rat brains, except that the
42-kDa band was by far the predominant band (not shown). We also used
antibody B825, raised against a peptide equivalent to amino acids
114-133 specific for G
11. This antibody is
selective for G
11, because it binds weakly to
recombinant G
q (13). Reactivity of B825
against 42-kDa proteins was barely detectable in sublingual membranes
but very intense in brain membranes (not shown). Antisera raised
against carboxy-terminal sequences specific for G
12
(AS233) and G
13 (AS343) each reacted with proteins in
sublingual membranes that displayed a relative mobility of ~43 kDa
(Fig. 8B, lanes 1 and 3,
respectively). Similar results were obtained with antibodies S20 and
A20, which were raised against amino-terminal domains of
G
12 and G
13, respectively (not shown).
Antisera CT92 (1), specific for G
14,
recognized a protein in brain but displayed no immunoreactivity to
sublingual membrane proteins of appropriate mass (Fig. 8C,
lanes 1 and 2, respectively).
|
Our results from Western analyses demonstrate the presence of
Gq, G
11, G
12, and
G
13 in sublingual membranes and, thus, further suggest
that one or more of these proteins may function in coupling to
muscarinic receptors. As an approach to distinguish G proteins coupled
directly to sublingual muscarinic receptors, we attempted to
immunoprecipitate specific G protein
-subunits from membranes after
photoaffinity labeling with [32P]GTP-azidoaniline in
either the presence or the absence of carbachol. As shown in Fig.
9A, lane 2,
radiolabeled proteins of ~42 kDa were detected in membranes
challenged with carbachol, and proteins were subsequently
immunoprecipitated with antibody Z811. No radiolabeled proteins were
detected in membranes not exposed to carbachol and/or in membranes
immunoprecipitated in the presence of antigen peptide (Fig.
9A, lanes 1, 3, and 4). These results
suggest that G
q and/or G
11 couple to
sublingual muscarinic receptors. To discriminate between these two G
proteins, we used antibodies E17 (G
q specific) and D17
(G
11 specific) in further experiments. Antibody E17 was used rather than antiserum WO82; the latter was raised against an
internal sequence of G
q and was previously demonstrated
not to immunoprecipitate native G
q proteins
(12). Furthermore, we confirmed the specificity of each
antibody by Western analysis in a preliminary experiment. No signal was
detected for either antibody when tested against 50 ng of the
alternative recombinant
-subunit protein, although positive controls
(5 ng of appropriate recombinant protein, 50 µg each of sublingual
membrane and brain membrane proteins) displayed strong signals (not
shown). In subsequent immunoprecipitation experiments, antibody E17
produced results similar to those of antibody Z811 (Fig.
9B). When antibody D17 was used with membranes treated
without carbachol (Fig. 9C, lane 1), there was a
diffuse band barely apparent at ~42 kDa, as were bands at positions
of higher and lower mass. In the presence of carbachol, only the 42-kDa
protein band displayed enhanced radiolabeling (Fig. 9C,
lane 2). Radiolabeling of 42-kDa proteins and most of the
higher and lower mass proteins was blocked by inclusion of the
G
11 antigen peptide whether membranes were challenged
with carbachol or not (Fig. 9C, lanes 3 and
4). These results indicated both G
q and
G
11 couple to muscarinic receptors in sublingual membranes. The proteins recognized by antibody D17 of mass greater and
less than 42 kDa likely represent unknown cross-reactive guanine nucleotide binding proteins, because similar proteins were detected in
Western analysis (Fig. 8A, lane 5) and after
photoaffinity labeling with [32P]GTP-azidoaniline (Fig.
6A, lanes 1-4). Using antibodies A20 (G
13 specific) or S20 (G
12 specific), we
were unable to detect radiolabeled proteins in immunoprecipitates from
membranes incubated either with or without carbachol (not shown). As
controls for these experiments, we demonstrated carbachol-enhanced
radiolabeling of 42-kDa proteins in separate membrane aliquots that
were subjected to SDS-PAGE and autoradiography immediately after
radiolabeling (not shown). In addition, we confirmed that antibodies
A20 and S20 immunoprecipitated G
13 and
G
12, respectively, by performing Western analysis of
immunoprecipitates using the same antibodies as probes. Thus muscarinic
receptors in sublingual membranes appear to couple primarily to
G
q and G
11. We considered conducting further experiments that would incorporate m1-toxin to distinguish the
relative coupling of G
q and G
11
to muscarinic M1 and M3 receptors in sublingual
membranes, but such experiments were determined to be unfeasible
because the immunoprecipitation experiments described above were near
the limits of detection; autoradiographic film required 4-6 wk of
exposure to indicate immunoprecipitated proteins.
|
To gain insight into the capacity of Gq relative
to that of G
11 to couple to sublingual muscarinic
receptors, we determined the relative abundance of each G protein
-subunit in sublingual membranes. We used Western analysis to
correlate the band intensities obtained with increasing amounts of
recombinant G
q (15-40 ng) and G
11
(8-24 ng) proteins with those obtained for two different amounts
of sublingual membrane proteins. Antibody D17 (200 ng/ml) was used to
probe for G
11. We used antiserum WO82 (1:1,000) rather than antibody E17 to probe for G
q because WO82 gave a
stronger signal and was in greater supply. As shown in Fig.
10, increasing amounts of
G
q and G
11 displayed linear distributions
of band intensities with respect to the amount of protein applied. The average (±SE, n = 4) amount of G
q in
sublingual membranes obtained in two separate experiments in which 15- and 25-µg membrane proteins were used was 1.2 ± 0.2 ng/µg
membrane protein. The value derived for G
11 in two
separate experiments in which 50- and 75-µg membrane proteins were
used was 0.21 ± 0.03 ng/µg membrane protein (mean ± SE,
n = 4). The abundance of G
q in
sublingual membranes is therefore nearly sixfold greater than that of
G
11.
|
Biochemical studies with cell lines have documented the efficient
coupling of M1 and M3 receptors to both
Gq and G
11 with the resultant
activation of PLC-
isoforms (8, 13). Accordingly, we
have found that inhibition of PLC blocks muscarinic activation of
exocrine secretion by sublingual mucous acinar cells (unpublished observations). Given that muscarinic receptors in sublingual membranes couple predominantly to G
q and G
11, we
used antibody Z811 to evaluate the direct role of these
-subunits in
the muscarinic activated hydrolysis of sublingual membrane
PIP2. In preliminary experiments, PIP2
hydrolysis was activated by GTP
S in a dose-dependent manner with
near-maximal activity (200% above basal level) at 100 µM GTP
S
(not shown). Carbachol (1 mM) alone had no effect on PIP2
hydrolysis (basal: 80.6 ± 5.2 pmol; carbachol: 82.6 ± 6.5 pmol; n = 7, P = 0.544). GTP
S was
required for carbachol activation of PIP2 hydrolysis, with
optimal activation at 1 µM GTP
S. Under these conditions, carbachol
increased PIP2 hydrolysis an average of 28% above that
observed by 1 µM GTP
S alone (carbachol + GTP
S: 127 ± 9.5 pmol; GTP
S: 99.2 ± 7.1 pmol; n = 7, P = 0.002). In subsequent experiments incorporating
antibody Z811 (Fig. 11A), we
found the antibody blocked stimulation of PIP2 hydrolysis
by 1 µM GTP
S, either with or without carbachol. The antibody was without these affects if first preincubated with its antigen peptide (Fig. 11A). As an additional control, nonimmune rabbit IgG
had no affects on PIP2 hydrolysis stimulated by either
GTP
S or carbachol plus GTP
S (Fig. 11A). In further
experiments, we found that specific inhibition of M1
receptors attenuates carbachol-induced PIP2 hydrolysis. As
shown in Fig. 11B, PIP2 hydrolysis induced by 1 mM carbachol was reduced nearly 60% by m1-toxin compared with control.
|
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DISCUSSION |
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---|
We determined the coupling of muscarinic receptors in rat
sublingual gland membranes to specific G protein -subunits. Coupling was initially verified by the GTP-induced shift in carbachol
high-affinity binding and by high-affinity GTPase activity. In both
cases, the effects of carbachol were insensitive to prior treatment of
membranes with PTX, although PTX catalyzed the ADP ribosylation of
membrane proteins of molecular mass consistent with that of
G
i and G
o subunits. In a similar manner,
CTX catalyzed the ADP ribosylation of 44-kDa membrane proteins
(presumably G
s) but had no effect on subsequent
carbachol-induced GTPase activity. Therefore,
-subunits susceptible
to ADP ribosylation by either PTX or CTX do not appear to regulate
directly muscarinic receptor-induced cellular functions in sublingual
glands. Toxin-sensitive G proteins in sublingual membranes may instead
mediate other receptors within sublingual glands such as vasoactive
intestinal peptide receptors and
2-adrenergic receptors,
presumably coupled to CTX-sensitive G
s and PTX-sensitive G
i, respectively (3).
Photoaffinity labeling of sublingual membrane proteins with
[32P]GTP-azidoaniline detected coupling of muscarinic
receptors to 42-kDa G protein -subunits. As determined by Western
analysis, G
q and G
11 in sublingual
membranes displayed the same electrophoretic mobility as the 42-kDa
proteins identified by carbachol-enhanced photoaffinity labeling. In
addition, antibodies specific for G
q or
G
11 immunoprecipitated 42-kDa proteins with enhanced
radioactivity after membranes were photoaffinity labeled in the
presence of carbachol. These results suggest sublingual muscarinic
receptors are coupled predominately to Gq and
G11.
The absence of G14, as assessed by Western analysis of
sublingual membranes, was not anticipated because G
14 is
expressed in other epithelial and glandular tissues, such as lung,
kidney, thymus, and testis (28). Both G
12
and G
13 were detected in sublingual membranes, although
the electrophoretic mobility of each
-subunit was slower than the
that of the 42-kDa proteins recognized by photoaffinity labeling.
Moreover, we were unable to immunoprecipitate detectable amounts of
radiolabeled G
12 and G
13 after
photoaffinity labeling of membranes in the presence or absence of
carbachol. Although we cannot rule out the possibility that these
latter negative results reflect the limited sensitivity of the
immunoprecipitation assay with native tissue, it appears that neither
G
12 nor G
13 couples significantly to
sublingual muscarinic receptors. Correspondingly, both
G
12 and G
13 have only been shown to
couple to nonmuscarinic receptors including thrombin, thromboxane
A2, bradykinin, and lysophosphatidic acid receptors. In
addition, both
-subunits regulate cellular events other than the PLC
or adenylyl cyclase pathways, such as Na+/H+
exchange and the c-Jun mitogenic pathway (7). The direct
effector(s) of G
12 and G
13 signaling have
yet to be established, although recent evidence suggests a role for
guanine nucleotide exchange factors (18). Further studies
of sublingual acinar cells are thus needed to elucidate the functional
role of G
12 and G
13.
In sublingual glands, muscarinic receptor activation results in the
generation of inositol 1,4,5-trisphosphate (30) and calcium-dependent fluid secretion (20). Furthermore,
recent results from our laboratory indicate that muscarinic-induced
exocrine secretion is also calcium dependent and is mediated by
activation of PLC (unpublished observations). In the present
investigation, we confirm the muscarinic stimulation of
PIP2 hydrolysis in sublingual membranes. More importantly,
Z811 (Gq/11 specific antibody) completely blocked this
response, suggesting that activation of PLC activity in sublingual
membranes by muscarinic receptors is mediated predominantly, if not
totally, by G
q and G
11. The absence of
muscarinic receptor coupling to PTX-sensitive G proteins (i.e.,
Gi proteins) in sublingual glands further argues against a
role for G protein
-subunits in the muscarinic activation of PLC.
A confounding factor in elucidation of muscarinic regulation of
exocrine secretion by sublingual mucous acini is the expression of
approximately equivalent amounts of M1 and M3
receptor subtypes (4). Moreover, immunolocalization
experiments indicate the coexpression of both M1 and
M3 receptor subtypes on sublingual mucous acinar cells
(unpublished observations). Blockade of M1 receptors by
m1-toxin results in a 40-50% decrease in the maximal muscarinic
receptor-induced exocrine response (5). Although m1-toxin
reduces the maximal secretory response, there is no appreciable change
in the apparent EC50 for agonist-induced secretion
(5). In the present study, we found that m1-toxin inhibits
approximately one-half of the carbachol-enhanced photoaffinity labeling
of 42-kDa G protein -subunits as well as the muscarinic activation
of PLC activity. Collectively, these results are consistent with
equivalent amounts of M1 and M3 receptors
expressed by acinar mucous cells that function separately to regulate
exocrine secretion through G
q/11 activation of PLC. A
maximal muscarinic receptor-induced secretory response requires
activation of both receptor subtypes.
Carbachol-enhanced photoaffinity labeling of 42-kDa proteins was
half-maximal (EC50) between 1 and 10 µM carbachol (Fig.
6B), similar to the EC50 (~7 µM) for
carbachol-stimulated GTPase activity. The high-affinity agonist binding
site of sublingual muscarinic receptors (Ki of
carbachol ~5 µM) thus mediates receptor coupling to G
proteins. In contrast, the EC50 reported previously for
carbachol-stimulated exocrine secretion by acinar cells is 0.3 µM
(3), ~10-fold lower than for receptor-G protein
coupling. This discrepancy in agonist affinities may reflect either an
abundance of muscarinic receptors or their associated G proteins linked
to exocrine secretion within acinar mucous cells. M3
receptors are not in excess, because secretion induced by these
receptors alone (after m1-toxin blockade of M1 receptors)
are insufficient to induce a maximal secretory response. Because the
number of M1 receptors expressed in isolated acini is
similar to that for M3 receptors, and M1
receptors are required for at least half of the maximal muscarinic
secretory response, we speculate they are also not in great excess. On
the other hand, the density of muscarinic receptors in sublingual gland
membranes is 462 fmol/mg protein (4). Based on our
estimates from Fig. 10 for the levels of Gq (28.6 pmol/mg protein) and G
11 (5.0 pmol/mg protein) in
membranes, the calculated molar ratio for each
-subunit relative to
the total pool of muscarinic receptors is 62:1 for G
q
and 11:1 for G
11. These ratios are doubled if one only
considers each receptor subtype separately given that approximately
equivalent amounts of M1 and M3 receptors are
expressed in sublingual membranes (4). These calculations
suggest both G
q and G
11 are in abundance
relative to muscarinic receptors, which may thus account, in part, for
the ~10-fold lower EC50 for carbachol-stimulated exocrine
secretion compared with receptor-G protein coupling. The nearly sixfold
higher expression of G
q relative to G
11
further suggests that Gq rather than G11
functions more appreciably in the coupling of sublingual M1
and M3 receptors to downstream effectors. Of course, it is
assumed these estimates are indeed representative of the pools of
-subunits functionally interactive with each receptor subtype in
mucous cells.
With respect to mucous cell exocrine function, it is unclear why both
M1 and M3 receptor subtypes serve to regulate
secretion in an apparently redundant manner (i.e., coupled to
Gq/11 and activation of PLC) and whether both receptors
are required to elicit a maximal secretory response. One explanation
may be that each receptor subtype and its corresponding downstream
elements are specifically compartmentalized, serving to form a
segregated intracellular signaling network (11). As an
example, a comparison of native vs. expressed exogenous muscarinic
receptors in Xenopus oocytes suggests coupling to distinct
calcium pools (15). There is also evidence that activation
of the PLC pathway may potentially modulate phospholipase D or
phospholipase A2 (8). One may thus speculate
that sublingual M1 and M3 receptors are
spatially segregated and linked to different pools of
G
q/11, PLC isozymes, and possibly other signaling
pathways to regulate distinct mucous cell functions other than
secretion, such as cell metabolism or gene expression. Clarification
must await delineation of the subcellular localization of
M1 and M3 receptors and their associated
networks of downstream signaling components in mucous cells.
Our results further accentuate differences among mammalian salivary
exocrine acinar cell types. Muscarinic activation of PLC and subsequent
intracellular calcium mobilization is the major pathway eliciting fluid
secretion in all three major salivary glands: parotid, submandibular,
and sublingual glands (2). In contrast with sublingual
glands, exocrine secretion by serous acinar cells of parotid glands and
seromucous acinar cells of submandibular glands are both regulated
primarily via -adrenergic receptors and the cAMP pathway
(2). Furthermore, M3 receptors are by far the
predominant muscarinic receptor subtype in parotid glands (6,
26). In rat parotid glands, M3 receptors are coupled to both G
q and G
i1 (6).
Also, parotid M3 receptors share an apparent redundancy
with
1-adrenergic receptors to mediate activation of PLC
via G
q/11 (24), whereas in rat sublingual glands,
1-adrenergic receptors are not expressed
(19). Signaling events regulating specific cell functions
are therefore not necessarily ubiquitous for the discrete exocrine cell
types that may be present, either together or independently, within the
numerous and diverse salivary glands lining the oral cavity. An
understanding of such differences is required if fully effective
therapeutic treatments are to be developed to treat those patients
suffering from hyposalivary function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Alan V. Smrcka for assistance in setting up the PIP2-hydrolysis assays and for helpful discussions.
![]() |
FOOTNOTES |
---|
This study was supported by National Institute of Dental Research Grant DE-10480.
Present address of W. Luo: Department of Cell and Cancer Biology, Division of Clinical Sciences, National Cancer Institute/National Institutes of Health, Bldg. 10, Rm. 3B47, 9000 Rockville Pike, Bethesda, MD 20892.
Address for reprint requests and other correspondence: D. J. Culp, Center for Oral Biology, 601 Elmwood Ave., Box 611, Rochester, NY 14642-8611 (E-mail: david_culp{at}urmc.rochester.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 2 May 2000; accepted in final form 30 October 2000.
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