Muscarinic acetylcholinereceptors regulating cellcycle progression areexpressed in human gingivalkeratinocytes

J. Arredondo1*, L. L. Hall1*,A. Ndoye1, A. I. Chernyavsky1,D. L. Jolkovsky2, S. A. Grando11Department of Dermatology, School ofMedicine, University of California, Davis,California, and 2Section of Periodontics, Schoolof Dentistry, University of California, LosAngeles, California, USA

It has long been reported and accepted

that acetylcholine (ACh) is involved in

most if not all aspects of basic neuro-

nal function. Recent experimental evi-

dence has suggested that ACh is not

restricted to neuronal cells, but is

widely expressed in pro- and eukary-

otic non-neuronal cells (1–3). Human

epidermal keratinocytes have been

identified as members of the growing

list of non-neuronal signaling networks

mediating intercellular communication

in which the cytotransmitter ACh acts

as a local hormone or a cytokine (4).

The effects of ACh are primarily

mediated via two classes of cholinergic

receptors, the nicotinic ACh receptors

(nAChR) and the muscarinic ACh

receptors (mAChR). We have recently

identified nAChRs expressed in human

attached gingival keratinocytes (GKC)

and characterized the cholinergic

enzymes that control levels of free ACh

Arredondo J, Hall LL, Ndoye A, Chernyavsky AI, Jolkovsky DL, Grando SA.

Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in

human gingival keratinocytes. J Periodont Res 2003; 38; 79–89. � Blackwell

Munksgaard, 2003

We have previously reported the presence in human gingival keratinocytes (GKC)

of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme,

acetylcholinesterase, the ACh degrading enzyme, and a3, a5, a7, b2 as well as a9nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh

in oral biology, we investigated the presence of the muscarinic ACh receptor

(mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4,

and m5 mRNA transcripts. Synthesis of the respective proteins was verified by

immunoblotting with the subtype-specific antibodies that revealed receptor bands

at the expected molecular weights. The antibodies mapped mAChR subtypes in

the epithelium of human attached gingiva and also visualized them on the cell

membrane of cultured GKC. The whole cell radioligand binding assay revealed

that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl

benzilate, Bmax ¼ 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream

coupling of the mAChRs to regulation of cell cycle progression in GKC was

studied using quantitative RT-PCR and immunoblotting assays. Incubation of

GKC for 24 h with 10 lM muscarine increased relative amounts of Ki-67, PCNA

and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were

abolished in the presence of 50 lM atropine. The finding in GKC of mAChRs

coupled to regulation of the cell cycle progression demonstrate further the struc-

ture/function of the non-neuronal cholinergic system operating in human oral

epithelium. The results obtained in this study help clarify the role for keratinocyte

ACh axis in the physiologic control of oral gingival homeostasis.

Sergei A. Grando, MD, PhD, DSc,Department of Dermatology, University ofCalifornia Davis Medical Center, 4860 Y Street,Suite #3400, Sacramento, California 95817,USATel: + 1 (916) 734 6057Fax: + 1 (916) 734 6793e-mail: sagrando@ucdavis.edu

Key words: muscarinic acetylcholine receptorsubtypes; human gingival keratinocytes; cellcycle progression regulators; muscarinic drugs

Accepted for publication November 5, 2001

*Drs Arredondo and Hall contributed to this

work equally. The order of first and second

authors is alphabetical.

J Periodont Res 2003; 38; 79–89Printed in the UK. All rights reserved

Copyright � Blackwell Munksgaard Ltd

JOURNAL OF PERIODONTAL RESEARCH

ISSN 0022-3484

in oral mucosa (5, 6). There exists

overwhelming evidence for the in-

volvement of keratinocyte ACh recep-

tors in the regulation of cell motility,

growth, and differentiation (7). Results

of studies with epidermal keratinocytes

suggested that mAChR activation

effects cell attachment, spreading, and

lateral migration (8). To identify a

more complete repertoire of the cho-

linergic molecules comprising the ACh

signaling axis in the oral epithelium

and to further explore the physiologic

role of this regulatory pathway, in this

study we investigated the presence,

structure and function of mAChRs in

GKC.

We report herein the identity of the

mAChR molecular subtypes present in

the human gingival epithelium and

cultured GKC. Results of RT-PCR

experiments, demonstrating predomi-

nant expression of m2, m3, m4, and

m5, were further verified in the immu-

noblotting and indirect immunofluo-

rescence (IIF) assays using specific

antibodies to each mAChR molecular

subtype protein. The radioligand

binding demonstrated that the recep-

tors are functional. The studies of

downstream effects of activation of

mAChRs in cultured GKC revealed

considerable changes in mRNA and

protein concentrations of the cell

cycle progression regulators. Taken

together, these results indicate that the

ACh axis of GKC includes functional

mAChRs coupled to regulation of the

cell cycle progression.

Materials and methods

Cell and tissue source

Fresh samples of normal human

attached gingiva were obtained from

periodontal surgical procedures (this

studywas approved by theUniversity of

California Davis Human Subjects

Review Committee). Attached gingival

samples destined for starting primary

cultures of GKC were freed of clotted

blood, and rinsed in Ca2+- and Mg2+-

free phosphate-buffered saline (PBS;

Gibco BRL, Gaithersburg, MD, USA).

Each sample was then cut into 3–4 mm

pieces, placed epithelium up into a

sterile cell culture dish containing 2.5 ml

of 0.125% trypsin (Sigma Chemical

Co., St. Louis,MO,USA) and 2.5 ml of

Minimum Essential Medium (Gibco

BRL) supplemented with 50 lg/mlgentamicin, 50 lg/ml kanamycin sul-

fate, 10 U/ml penicillin G, 10 lg/mlstreptomycin, and 5 lg/ml amphoteri-cin (all from Gibco BRL). Individual

tissues were incubated overnight at

37�C in a humidified atmosphere with

5%CO2. The epithelial sheets were then

separated from the lamina propria in

Minimum Essential Medium contain-

ing 20% heat inactivated newborn calf

serum (Gibco BRL), and individual

GKC were isolated by gentle pipetting

followed by centrifugation, as detailed

elsewhere (5). Cultures of GKC were

grown at 37�Cand 5%CO2 in 25 cm2 or

75 cm2 Falcon culture flasks (Corning

Glass Works, Corning, NY, USA) in

serum-free keratinocyte growth

medium (KGM; Gibco BRL) contain-

ing 0.09 mM Ca2+. KGM was changed

every 3 days, and cultures were pas-

saged upon reaching approximately

80% confluence. For cell exposure

experiments, both the pan-muscarinic

agonist muscarine chloride and the pan-

muscarinic antagonist atropine were

purchased from Sigma Chemical Co.

RT-PCR assay

Total RNA was extracted from cul-

tured human GKC using guanidinium

thiocyanate phenol chloroform extrac-

tion procedure (TRIzol Reagent, Gibco

BRL), as described elsewhere (9). The

quantity and structural integrity of

RNA samples was confirmed by elec-

trophoresis on 2% agarose/2.2 M

formaldehyde gels, and by optical

density of the 260/280 nm ratio. Only

samples that showed intact 28S and 18S

ribosomal RNA bands and exhibited a

260/280 nm ratio of >1.8 were used in

the experiments. One microgram of

dried, DNase-treated RNA was reverse

transcribed in 20 ll of RT-PCR mix

[50 mM Tris (pH 8.3), 6 mM MgCl2,

40 mM KCl, 25 ll dNTPs, 1 lg Oligo-

dt (Gibco BRL), 1 mM DTT, 1 U

RNase inhibitor (Boehringer, Mann-

heim, Germany) and 10 U SuperScript

II (Gibco, BRL)] at 42�C for 2 h. The

PCR was carried out in a final volume

of 50 ll containing 2 ll of the single

strand cDNA product, 10 mM Tris-

HCl (pH 9.0), 5 mM KCl, 5 mM

MgCl2, 0.2 mM dATP, 0.2 mM dCTP,

0.2 mM dGTP, 0.2 mM dTTP and

2.5 U Taq DNA polymerase (Perkin

Elmer, San Jose, CA, USA) and 20 pM

each of both the sense and the antisense

primers. To allow the quantitative

determination of relative gene expres-

sion levels, the cDNA content of the

samples was normalized, and the linear

range of amplification was determined

for each primer set. Then, samples from

each drug-treated (experiment) and

control (not treated) GKC were am-

plified using sets of primers shown in

Table 1. In each experiment, the

housekeeping gene glyceraldehyde-

3-phosphate dehydrogenase (GAPDH)

was amplified concurrently with each

particular gene of interest. The cycling

was performed at 94�C (1 min), 60�C(2 min), and 72�C (3 min) for 24–30

cycles. The PCR products from

experimental and control samples were

run in parallel on a 2% Sea Kem LE

agarose gel (FMC, Riceland, ME,

USA), stained with ethidium bromide,

and the gels were then scanned using

Alpha Imager 2000 (Alpha Innochet,

Inc., San Leandro, CA, USA). In each

experiment, the relative gene expression

level was determined based on the

density of the relevant DNA band. To

standardize results, the mean density

value of each band was expressed rela-

tive to the control value. The control

value for the expression of each par-

ticular gene was determined by mea-

suring the intensity of its cDNA band

in non-treated, parallel cultures and

taken as 1. Negative controls included:

(a) omission of the RT step; and (b)

blank samples consisting of reaction

mixtures without RNA, both of which

were run together with experimental

samples.

Immunoblotting assay

Proteins were isolated from the phenol-

ethanol supernatant by adding 1.5 ml of

isopropyl alcohol per 1 ml of TRIzol

Reagent used for the initial homogeni-

zation of cultured GKC. Protein pellets

were washed three times with 2 ml of

0.3 M guanidine hydrochloride in 95%

ethanol, and then one time with 2 ml of

80 Arredondo et al.

95% ethanol. The pellets were dissolved

in sample application buffer [1.0 ml of

0.5 M Tris-HCl (pH 6.8), 1.9 g ultra

pure urea (Fisher Scientific, Tustin, CA,

USA) and 10%SDS (Fisher Scientific)].

Proteins were separated in 15% SDS-

PAGE, electroblotted onto an 0.2-lmnitrocellulose membrane (Bio-Rad,

Hercules, CA, USA), and blocked

overnight at 4�C in the blocking buffer

consisting of 5% non-fat dried milk in

0.1% (v/v) Tween 20 (Sigma Chemical

Co.), 25 mM Tris-HCl (pH 8), 125 mM

NaCl and 0.05% sodium azide. In each

triplicate assay, the experimental and

control samples were run on the same

gel. The primary antibodies were dilut-

ed in the blocking buffer and incubated

for 1 h at room temperature. The spec-

ificity and the dilutions of primary

antibodies used, and their sources are

listed in Table 2. The secondary anti-

bodies [sheep antimouse or donkey

antirabbit Ig labeled with HRP

(Amersham Pharmacia Biotech, Inc.,

Piscataway, NJ, USA)] were diluted

1 : 3000 in the blocking buffer lacking

sodium azide and applied to the mem-

brane for 1 h at room temperature. The

membranes were developed using the

ECL + Plus chemiluminescent detec-

tion system (Amersham Pharmacia

Biotech, Inc.). To visualize antibody

binding, the membranes were scanned

with FluorImager/Storm� (Molecular

Dynamics, Mountain View, CA, USA),

and the intensity of bands was analyzed

using the ImageQuant software

(MolecularDynamics). The results were

standardized by expressing the density

of each protein band under investiga-

tion in the experimental (i.e. a muscar-

inic drug-treated GKC) sample relative

to the value determined in the control

(i.e. intact GKC) sample taken as 1. The

specificity of staining was controlled

in negative control experiments, in

which the antiserum was preincubated

with the specific peptide used for

immunization or the primary antibody

was either omitted or replaced with an

irrelevant, isotype- and species-match-

ing antibody.

IIF assay

The mAChR subtypes were visualized

in the attached gingival tissue and

GKC cultures using the rabbit polycl-

onal antireceptor antibodies that were

characterized and used by us in the

past (10–12) and are now commercially

available from Research & Diagnostic

Antibodies (Benicia, CA, USA).

Freshly obtained samples of human

attached gingiva were cut in pieces and

fixed for 3 min with 3% fresh depoly-

merized paraformaldehyde that con-

tained 7% sucrose, to avoid cell

permeabilization, and stored frozen at

)75�C until sectioned. On the day of

experiments, the tissue specimens or

coverslips with similarly fixed 2nd

passage GKC were washed with PBS

and incubated overnight at 4�C with a

primary anti-mAChR subtype-specific

antibody diluted 1 : 1000 in PBS. After

washing, the slides were exposed for

1 h at room temperature to fluorescein

isothiocyanate (FITC)-conjugated

swine antirabbit IgG antibody (DAKO

Corporation, Carpenteria, CA, USA)

diluted 1 : 30 in PBS. The specimens

were examined with an Axiovert 135

fluorescence microscope (Carl Zeiss

Inc., Thornwood, NY, USA). The

specificity of antibody binding in IIF

experiments was demonstrated by

omitting the primary antibody or by

replacing primary antibody with an

irrelevant antibody of the same isotype

and species as the primary antibody.

Table 1. The human mAChR subunit and cell cycle genes studied by RT-PCR

Name Abbreviation Gene name Accession no. Primers

Glyceraldehyde-3-phosphate dehydrogenase GAPDH GADP J04038 214–234, 401–449

mAChR:

subtype m1 m1 CHRM1 XM006058 375–394, 840–863

subtype m2 m2 CHRM2 XM004724 433–456, 868–892

subtype m3 m3 CHRM3 U40583 367–388, 814–835

subtype m4 m4 CHRM4 XM006296 1302–1326, 1747–1770

subtype m5 m5 CHRM5 XM012454 1271–1296, 1561–1584

p53-dependent G2 arrest p53 REPRIMO AB04385 475–496, 820–839

Cdk inhibitor p21 binding protein 1 p21 TOK-1 AB040450 319–340, 601–623

Proliferation-related Ki-67 antigen Ki-67 MKI67 X65550 299–321, 727–750

Proliferation cell nuclear antigen PCNA PCNA M15796 236–259, 535–555

Cyclin D1 Cyl 1 CCND1 M64349 279–301, 568–589

Table 2. The primary antibodies used in this study

Antibody Isotype Host

Dilution

(lg/ml) Epitope Reactivity

mAChRm1a IgG rabbit 1 PLMAREDA human and rodents

mAChRm2a IgG rabbit 1 PVHIGNANK human and rodents

mAChRm3a IgG rabbit 1 FVEAVSKDFA human and rodents

mAChRm4a IgG rabbit 1 HSDDHSAPSSK human and rodents

mAChRm5a IgG rabbit 1 EGPYAAORD human and rodents

p53b IgG1 mouse 5 RHSVV human and rodents

p21b IgG1 mouse 1 TSMTDFYHSKRR human and rodents

Ki-67b IgG1 mouse 1 2597–2896 human and rodents

Cyclin D1b IgG2 mouse 1 1–295 (whole protein) human and rodents

PCNAb IgG2 mouse 2.5 1–261 (whole protein human and rodents

aResearch and Diagnostic, Berkeley, CA, USA.bOncogene Research Products, Boston, MA, USA.

Gingival keratinocyte muscarinic receptors 81

Whole-cell radioligand bindingassay

Binding experiments were performed

with the pan-muscarinic radioligand

[3H]quinuclidinyl benzilate ([3H]QNB)

using a modification of the whole-cell

radioligand binding assay detailed

elsewhere (13). Briefly, 2nd passage

GKC were loaded into 24-well plates

and grown as monolayers in KGM to

�80% confluence. On the day of

experiment, the monolayers were

washed with ice-cold PBS at 4�C for

15 min, put on ice, and exposed in

triplicate for 60 min to from 0.01 to

100 nM concentrations of [3H]QNB

(43 Ci/mmol; NEN, Boston, MA,

USA) with gentle shaking. Non-specific

binding was measured in parallel wells,

in which GKC were exposed to

[3H]QNB in the presence of the 100 lMconcentration of non-labeled atropine.

Following the incubation, the mono-

layers were washed four times with

ice-cold PBS to remove unbound

[3H]QNB, solubilized in 200 ll of 1%SDS, and the radioactivity was counted

in a liquid scintillation counter. To

determine the cell number per well,

the GKC harvested from randomly

selected wells were counted in a hemo-

cytometer. Saturation isotherms were

analyzed according to a model of one-

site binding, and the binding capacity

(Bmax) and dissociation constant (Kd)

were calculated using the non-linear

regression analysis program Prism

(Graph-Pad Software, San Diego, CA).

Statistical analysis

The experiments were performed in

triplicates. The results of quantitative

assays were analyzed to obtain mean ±

SD. Statistical significance was calcu-

lated using Student’s test, andP <0.05

indicated significant differences.

Results

Amplification by RT-PCR of themAChR subtype RNAs expressed inGKC

RT-PCR was performed using mAChR

subtype-specific primers previously

characterized by us (12) and total RNA

extracted from the 2nd passage cultures

of GKC grown to �80% confluence in

KGMcontaining 0.09 mM extracellular

Ca2+. We routinely detected, in all

samples, amplification sequences for

mRNAs encoding m2, m3, m4, and m5

(Fig. 1A; lanes 1, 6, 11 and 16), whereas

m1 was only detected in one of three

samples (data not shown). The ability of

the primers to amplify the specific

sequences was confirmed in positive

control experiments in which the

amplification of each receptor subtype

from genomic DNA, possible due to the

absence of introns, produced detectable

band on an electrophoresis gel (Fig. 1A;

lanes 2, 7, 12 and 17). PCRof aliquots of

total RNA from each sample following

DNase treatment and subsequent RT,

without the addition of the reverse

transcriptase, produced no detectable

product, confirming that the products

were amplified fromRNA and not from

putative DNA, which possibly might

contaminate RNA samples (Fig. 1A).

Each band of interest amplified from

RNA was found to be of the expected

size, relative to the base pair marker

used, and amplification product

sequencing further verified that the

correct products had been amplified.

Identification by immunoblottingof the mAChR subtype proteinsexpressed in GKC

Antibodies specific to m1, m2, m3, m4,

and m5 mAChR subtypes were used to

probe total cellular proteins of GKC

resolved by SDS-PAGE. As seen in

Fig. 1B, each of m2, m3, m4, and m5

antibodies uniquely and specifically

visualized a single major protein band

with an apparent molecular mass of

65 kDa (m2), 70 kDa (m3 and m4) and

95 kDa (m5), which corresponds to the

molecular masses of these mAChR

subtypes reported previously by us (10,

12, 13) and other workers (14–17). The

specific staining observed with these

mAChR subtype-selective antibodies

was abolished by preincubation with

the respective mAChR peptide used for

immunization (data not shown). Nei-

ther was any protein band observed in

other negative control experiments

in which the primary antibody was

omitted.

A weak staining produced by m1

antibody (not shown) was not ade-

quate to conclude that the m1 mAChR

protein is present in GKC in mean-

ingful quantities.

Visualization by IIF of the mAChRsubtypes expressed in the attachedgingiva and cultured GKC

The mAChR subtype-selective anti-

bodies were used in the IIF assays to

map receptor proteins in cryostat sec-

tions of normal human attached gin-

givas and the 2nd passage cultures of

GKC. In oral tissue samples, each of

m2, m3, m4, and m5 antibodies pro-

duced an intercellular, web-like stain-

ing of the epithelium (Fig. 2), which is

consistent with the antibody binding to

the cell membrane (18). The m2 anti-

body stained the entire mucosa, being

most abundant in the middle epithelial

compartment. In contrast, the bulk of

m3 and m4 immunoreactivities were

localized to the lowermost rows of the

epithelial cells, especially to the rete

ridges. The m5 antibody stained pre-

dominantly the lower 2/3 portion of

the epithelium.

In vitro, each of m2, m3, m4, and

m5 antibodies produced specific stain-

ing of individual GKC in preconfluent

cultures (Fig. 2). The immunostaining

originated from the cell membranes,

because the cells were not permeabi-

lized during fixation. The m2 antibody

produced a diffuse staining of the

entire cell surface, being most abun-

dant at the cell borders. The m3, m4,

and m5 antibodies decorated predom-

inantly the periphery of the cell mem-

brane, producing a distinct granular

staining pattern, which suggested that

these mAChR subtypes form clusters

on the cell surface of GKC. Individual

clusters were particularly apparent at

the cell borders. In confluent cultures,

the immunostaining acquired an inter-

cellular, fishnet-like pattern (e.g. m4 in

Fig. 2) characteristic of that seen in the

gingival tissue samples.

The IIF staining was abolished by

preincubating the specific antiserum

with the synthetic peptide used to

generate the antibody, and when the

primary antibody was omitted or

replaced with an isotype-matching

82 Arredondo et al.

irrelevant primary antibody control

(data not shown).

Ligand-binding properties ofmAChRs expressed by GKC

The mAChR expression level in GKC

and receptor affinity were character-

ized in whole-cell radioligand binding

assay using the reversible, lipophilic,

tertiary muscarinic antagonist

[3H]QNB. The results showed a satu-

rable specific binding of [3H]QNB to

the cell surfaces of 2nd passage GKC

(Fig. 3). Equilibrium binding experi-

ments identified displaceable binding

sites with a Kd of 62.95 pM and a Bmax

of 222.9 fmol/106 cells, corresponding

to approximately 1.3 · 105 sites/cell.

These kinetic parameters of the dis-

placeable [3H]QNB binding sites in

GKC are similar to the mAChR

binding sites identified in other types of

non-neuronal cells (13, 15, 19).

The mAChR-dependent regulationof the cell-cycle gene expressionin GKC

To characterize the biologic role of

mAChRs expressed in GKC, with

respect to their downstream effects on

relative mRNA and protein levels of the

well-known cell cycle regulators, we

used gene-specific RT-PCR primers

and monoclonal antibodies listed in

Tables 1 and 2, respectively. The

RT-PCR designed to amplify the

human cell cycle regulator genes p53,

p21, Ki-67, cyclin D1 and PCNA all

yielded products of expected sizes. As

seen in Fig. 4A, the GKC stimulated

with the pan-muscarinic agonist musc-

arine, 10 lM, for 24 h showed increased

levels of Ki-67 (2.8 fold), PCNA (1.5

fold) and p53 (1.7 fold) mRNA tran-

scripts, whereas the relative amounts of

mRNAs coding for p21, and cyclin D1

Fig. 1. Identification of the molecular subtypes of mAChRs expressed in cultured human GKC. Second passage normal human GKC were

used to extract total RNA and proteins for the RT-PCR and immunoblotting assays detailed in Materials and methods. A. The mAChR

subtype gene expression determined using subtype-specific primers (Table 1) and keratinocyte cDNA as a template. Each pair of primers

yielded a PCR product of the expected size: 348 bp for m2, 496 bp for m3, 430 bp for m4, and 397 bp for m5. Lanes 1, 6, 11 and 16 represent

DNase-treated RNA samples that were amplified by RT-PCR. Lanes 2, 7, 12 and 17 are positive controls wherein DNase treatment of RNA

samples was omitted so that the genomic DNA could be amplified. Lanes 3, 8, 13 and 18 are negative controls wherein the RT-PCR step was

omitted. Lanes 4, 9, 14 and 19 are negative controls wherein the cDNA template was omitted. Lanes 5, 10, 15 and 20 are the 100 bp DNA

ladder standard. The molecular weights of amplified bands are shown at the bottom of the gel. B. The keratinocyte mAChR subtype proteins

visualized in Western blots. Results of a representative experiment showing protein bands recognized by rabbit polyclonal antibodies specific

for m2, m3, m4, and m5 subtypes (Table 2) among total cell proteins of the 2nd passage normal human GKC resolved by 15% SDS-PAGE

and immunoblotted as described in Material and methods. The apparent molecular mass of each receptor protein is shown in kDa at the

bottom of the gel.

Gingival keratinocyte muscarinic receptors 83

remained unchanged. The GAPDH

gene amplification remained constant in

each experiment (Fig. 4A). The nega-

tive control experiments failed to pro-

duce any amplified product (data not

shown). These effects of muscarine were

completely blocked in the presence of

50 lM of the pan-muscarinic antagonist

atropine (Fig. 4A), indicating that the

Oral Mucosa Cell Culture

m2

m3

m4

m5

84 Arredondo et al.

observed alterations in the cell

cycle gene expression resulted from

the mAChR-mediated intracellular

signaling.

By immunoblotting, we also found

that the relative amounts of p53 and

PCNA, as well as p21 and cyclin D1

went up after the 24 h exposure to

muscarine (Fig. 4B). The relative

amounts of each up-regulated cell cycle

protein increased in a range from 1.6 to

2.1 fold. Atropine abolished the effects

of muscarine (Fig. 4B). Each protein

band was visualized at the expected

molecular weight, namely: cyclin D1 at

approximately 35 kDa (20), PCNA at

37 kDa (21), and p53 and p21 at 53

and 21 kDa, respectively. The protein

levels of Ki-67 in experimental and

control GKC did not differ signifi-

cantly (data not shown).

Taken together, the results of

RT-PCR and immunoblotting experi-

ments indicated that the expression of

PCNA and p53 genes in muscarine-

treated cells was up-regulated at both

the transcriptional and translational

levels. Expression of the Ki-67 gene

was up-regulated at the transcriptional

level and that of the p21 and cyclin D1

genes at the translational level only.

Discussion

In this study, we demonstrate for the

first time that normal human GKC

both in vivo and in vitro express the

m2, m3, m4, and m5 molecular sub-

types of the classic mAChRs. The

receptor molecules are abundantly

expressed on the cell membrane where

they can be bound by endogenously

produced and secreted ACh and cho-

linergic muscarinic drugs. The down-

stream signaling from these mAChRs

proceeds via a pathway that up-regu-

lates the expression of cell cycle pro-

gression regulators at both

transcriptional and translational lev-

els. These findings advance our

knowledge about the role of the ker-

atinocyte ACh axis in oral biology.

ACh is a ubiquitous chemical in life

best known for its role in neurotrans-

mission. Increasingly, a wider role for

ACh in various aspects of non-neuronal

cell functions is being recognized (1–4,

7, 22, 23). ACh is synthesized from

choline and acetyl coenzyme A in an

enzymatic reaction catalyzed by choline

acetyltransferase and hydrolyzed to

acetate and choline by acetylcholinest-

erase. Choline acetyltransferase and

two molecular forms, the asymmetric

and the globular forms, of acetylcho-

linesterase were found in the oral epi-

thelium using a combination of

molecular biological and immunohis-

tochemical assays (5). Free ACh has

been detected by an HPLC method

in the human oral epithelium (0.7–

8 pmol/sample) (22) as well as other

parts of the alimentary tract (24). There

is an upward concentration gradient of

free ACh in the mucosal epithelium (5).

ACh does not readily cross lipid

membranes because it is highly polar

and positively charged (25). ACh and

related compounds elicit biological

effects due to binding to nAChR and

mAChR. These two cholinergic recep-

tor classes may coexist in individual

cells and be functionally interrelated so

that the induction of one receptor class

affects, either positively or negatively,

the induction of the other (26–33). The

notion that ACh acts as a local hor-

mone in mucosal epithelium is sup-

ported by the fact that choline, the

metabolite of ACh, can activate cho-

linergic receptors after ACh has been

cleaved by acetylcholinesterase (34, 35).

The mAChRs are G protein-coupled

receptors that induce different intracel-

lular signaling in the cells in which they

are expressed (36–38). The mAChRs

can be grouped according to their

functionality. The m1, m3, and m5

molecular subtypes activate protein

Fig. 3. Specific binding of [3H]QNB to cultured human GKC. The specific binding of

[3H]QNB was achieved at 0�C in the monolayers of GKC in flat-bottomed 24-well plates

exposed to increasing concentrations of [3H]QNB in the absence (total binding) or presence

(non-specific binding) of 100 lM non-labeled atropine, as described in Material and methods.

The specific binding was computed by subtracting the non-specific binding from the total

binding.

Fig. 2. Visualization of mAChRs in human

attached gingiva and cultures of GKC.

Rabbit polyclonal antibodies raised to

unique protein sequences of m2, m3, m4,

and m5 mAChR subtypes were used to

probe cryostat sections of freshly frozen

specimens of normal human attached ging-

iva and 2nd passage cultures of normal

human GKC by IIF. Binding of the pri-

mary antibodies was visualized using sec-

ondary, FITC-labeled antirabbit IgG

antibody (see Materials and methods).

In gingiva, the subtype-selective antibodies

visualized their target receptor proteins on

the cell membrane of GKC, producing a

characteristic �fishnet�-like intercellular

staining. In cultures of GKC, the antibodies

visualized the mAChR subtypes as either

small (m2) or large (m3 and m5) distinct

bright dots decorating the immediate peri-

nuclear as well as more peripheral areas of

the cell membrane of individually located

GKC, and produced a typical intercellular

staining in the areas of complete confluence,

as shown for m4. Preincubation of the an-

tipeptide immune sera with the synthetic

peptides used for immunization, omitting

the primary antibody or replacing it with

an irrelevant antibody of the same iso-

type and species as the primary antibody

abolished the fluorescent staining. Scale

bars ¼ 20 lm.

Gingival keratinocyte muscarinic receptors 85

kinase C by elevating intracellular Ca2+

and diacylglycerol, whereas the m2

and m4 inhibit protein kinase A by

diminishing adenylye cyclase activity,

resulting in the reduction of intracellu-

lar levels of cyclic adenosine mono-

phosphate. The cells activated by ACh

amplify and further spread the signal by

releasing messengers such as calcium

A

B

Fig. 4. Alterations of the cell cycle gene

expression in GKC exposed to muscarine.

Second passage normal human GKC were

exposed to 10 lM muscarine in the absence

or presence of the specific muscarinic

antagonist atropine, 50 lM, for 24 h at 37�Cand 5% CO2. Control GKC were incubated

in the same KGM without any drugs. The

total RNA and proteins extracted from both

exposed and control cultures were subjected

to quantitative analysis of the cell cycle

regulators p21, p53, Ki-67, PCNA and

cyclin D1 as detailed in Materials and

methods. Approximately 2.5 · 106 viable

cells served as a source of RNA and protein

in each experimental condition. The images

represent typical appearance of the bands in

gels. The ratio data are the means of the

values obtained in at least three independent

experiments. A. The mRNA levels of the cell

cycle regulator genes determined by

RT-PCR using specific primers (Table 1). A

PCR product of the expected size was

amplified by each primer set specific for

p53 (389 bp), PCNA (307 bp), cyclin-D1

(482 bp), Ki-67 (499 bp), and p21 (233 bp).

Amplification of the GAPDH gene product

(354 bp) was used to normalize the cDNA

content in each sample, and as a positive

control for RT-PCR effectiveness. Exposure

to muscarine increased the p53, PCNA and

Ki-67 mRNAs levels by 1.7, 1.5 and 2.8 fold,

respectively. The presence of atropine abol-

ished these changes. B. Changes in the rela-

tive amounts of the cell cycle proteins p53,

p21, PCNA and cyclin-D1 analyzed by

Western blots. The picture shows results of a

representative experiment in which each

protein band was visualized by a specific

monoclonal antibody (Table 2) at the

expected molecular weight (shown in kDa to

the right of the gels). The relative amounts of

p53, p21, PCNA and cyclin D1 increased

several times, ranging from 1.6 to 2.1-fold, in

GKC exposed to muscarine given alone but

not to the same dose of muscarine in the

presence of atropine. The staining of the

protein bands was absent in the negative

control experiments in which the membranes

were treated without primary antibody, with

irrelevant primary antibody of the same

isotype and host, or the antipeptide antisera

prior to treatment of the blotting membrane

were preincubated with the specific peptide

used for immunization (not shown).

86 Arredondo et al.

ions, eicosanoids, nitric oxide, other

cytotransmitters, cytokines, growth

factors, and other biological effector

molecules. Activation of ACh receptors

can affect the functioning of motor

and structural proteins and integrin

receptors through cascade reactions

mediated by protein kinases. The pro-

tein kinase activities, which are known

to result from activation of ACh

receptors, include cGMP-dependent

protein kinase, calcium/calmodulin-de-

pendent protein kinase, protein kinase

C and protein tyrosine kinase (39).

In this study, the expression of the

genes coding for various molecular

subtypes of mAChRs in human gingi-

val epithelium was analyzed using

RNA and proteins isolated from cul-

tured human GKC. To identify the

profile of gingival keratinocyte

mAChRs, we used a combination of

molecular biological and immunohis-

tochemical approaches. Ligand-bind-

ing probes currently available do not

clearly distinguish among the subtypes,

due to their structural homology and

pharmacological similarity. We

screened keratinocyte RNA using

mAChR subtype-specific primers by

RT-PCR and consistently amplified

nucleotide sequences corresponding to

authentic m2, m3, m4, and m5, but not

to m1, mAChRs. The validity of PCR

amplification of mAChRs, coded by

the intronless genes, was illustrated in

negative control experiments in which

omission of the RT step abolished

amplification. In the past, the use of

our mAChR subtype-selective PCR

primers revealed the expression of dif-

ferent combinations of m1–m5

mRNAs in other types of mucocuta-

neous cells, such as epidermal kerati-

nocytes (m1, m3, m4, and m5) (10),

skin fibroblasts (m2, m4 and m5) (12),

and melanocytes (m1-m5) (40). Results

of the immunoblotting and IIF assays

further verified the mAChR subtypes

expressed by GKC in vivo and in vitro.

The antibodies specific to m2, m3, m4,

and m5 visualized these mAChRs

subtypes among keratinocyte proteins

resolved by SDS-PAGE, and also

produced an intercellular staining pat-

tern in normal human attached gingiva

consistent with the presence of receptor

molecules on the cell membrane of

GKC. The localization of mAChRs

subtypes in the gingival epithelium

appeared to be very similar to and

reminded that observed by us earlier in

the epidermis (10).

The repertoire of mAChR subtypes

expressed by GKC differs from that

found in epidermal keratinocytes by

the lack of m1 and the presence of m2.

The absence of m1 is not surprising

because expression of this mAChR in

the epidermis occurs at the latest stage

of differentiation (10), which does not

normally occur in mucosa. The pres-

ence in GKC of m2, which is also

abundantly expressed by the mesen-

chymal cells such as dermal fibroblasts

(12), is of interest since it may be

related to the differences in the

muscarinic pathway of the ACh-

dependent cell cycle regulation in

the oral vs. epidermal variants of the

stratified epithelium. In addition to the

mAChRs subtypes identified in this

study, the epithelial cells lining human

attached gingiva and the upper two-

third portion of the esophageal muco-

sa have been previously shown to

express other types of cholinergic

receptors mediating ACh signaling

(5, 6). These include a3, a5, a7, a9, b2,and b4 (not reported previously)

nAChR subunits assembling the ACh-

gated ion channels that regulate cell

adhesion and motility. Thus, GKC

respond to ACh via cholinergic recep-

tors of both nicotinic and muscarinic

classes, including a9, a first of its kind

AChR with dual, nicotinic-and-mu-

scarinic pharmacology that was cloned

from human GKC (6). Hence, the

physiologic control of GKC by ACh

can be mediated by two distinct types

of biochemical events: (i) the metabolic

events, elicited by ACh binding to the

G protein-coupled single-subunit

transmembrane glycoproteins, or

mAChRs; and (ii) the ionic events,

generated by opening of ACh-gated

ion channels represented by keratino-

cyte nAChRs. Simultaneous stimula-

tion of keratinocyte nAChRs and

mAChRs by endogenously secreted

ACh may be required to synchronize

and balance between ionic and meta-

bolic events in a single keratinocyte.

In this study, we determined that

stimulation of the muscarinic

pathways of ACh signaling in GKC

produces rapid and profound effects

on the expression of cell cycle regula-

tors. Muscarine produced a several

fold increase of Ki-67, a proliferation

marker expressed in the nucleolus

during G1, S, G2 and M phase (41),

PCNA (proliferating cell nuclear

antigen) an auxiliary factor for DNA

polymerase d and e that is expressed

primarily at G1-S phase and plays a

role in chromosomal DNA replication

and repair (42). Simultaneously with

up-regulation of these cell cycle pro-

gression-associated markers, stimula-

tion of mAChRs in GKC also led to a

reciprocal increase of p53 and p21,

which play a significant role in the

induction of cell cycle arrest at G1/S.

p53, known as a tumor suppressor

gene, regulates the transcription of

p21 thereby inhibiting S phase entry

primarily via inhibition of cyclin-

dependent protein kinases and p21

binding and inhibition of PCNA. p53

may also play a role in the induction

of apoptosis via both mitochondrial

and receptor mediated pathways, evi-

denced by increased expression of

caspases (43). These findings suggest

that downstream signalling from

mAChRs expressed in GKC initiates

complex changes in cell cycle regula-

tion, including proliferation-inducing

effects, DNA repair and replication

anomalies, and pro-apoptotic gene

activation. In the past, ACh acting via

its muscarinic pathways has been

demonstrated to control apoptosis

and exhibit growth factor-like effects,

including differential regulation of

immediate-early gene expression

(44–47). Therefore, a major biological

function of free ACh in oral mucosa

could be to coordinate the process of

keratinocyte development.

The muscarinic effects on the

expression of cell cycle regulators

observed in this study could be medi-

ated by several molecular subtypes of

mAChRs because both muscarine and

atropine can act upon all known

mAChR subtypes (48). Further studies

are needed to dissect the specific role(s)

of each mAChR subtype expressed in

GKC in the physiologic control of

these cells by ACh. In experiments with

human epidermal keratinocytes, we

Gingival keratinocyte muscarinic receptors 87

have previously observed that activa-

tion of m4 is required to sustain kera-

tinocyte migration (49). When this

pathway of ACh signaling was

blocked, the migration distance of

keratinocytes decreased. Furthermore,

blocking a muscarinic pathway with

propylbenzilylcholine mustard stimu-

lated keratinocyte proliferation (8),

which might result from inactivation of

an odd-numbered mAChR that inhib-

its cell proliferation (m3) (50) and

clonogenic potential (m5) (51).

In conclusion, it is evident from this

study and previously published ob-

servations (5) that the repertoire of

both cholinergic enzymes and both

classes of cholinergic receptors

changes during keratinocyte differen-

tiation. This phenomenon may have

biological meaning because it renders

each developmental stage a unique

combination of cholinergic signaling

molecules, which may allow the single

cytotransmitter ACh to exert differ-

ential effects on GKC at various

stages of their development in the

mucosal epithelium. In this model,

binding of ACh to the cell membrane

simultaneously elicits several diverse

biochemical events the �biological sum�of which, taken together with cumu-

lative effects of other hormonal and

environmental stimuli, determines a

distinct change in the cell cycle. Thus,

by simultaneously activating both

cholinergic receptor classes, ACh can

play a role of �pace maker� for kera-

tinocyte development. Therefore,

elucidation of a mAChR subtype-

selective control of the gene expres-

sion responsible for acquisition of a

particular cell phenotype in the course

of differentiation of GKC will provide

a mechanistic insight into a general

regulatory mechanism driving epithe-

lial turnover in the upper digestive

tract, and will also help focus future

mechanistic studies on either pre or

post-transcriptional events regulated

by each particular mAChR subtype.

Acknowledgements

This work was supported in part by the

research grant #0713 from the

Smokeless Tobacco Research Council,

Inc., New York, NY 10170 (to SAG).

References

1. Sastry BV, Sadavongvivad C. Cholinergic

systems in non-nervous tissues. Pharmacol

Rev 1978;30:65–132.

2. Wessler I, Kirkpatrick CJ, Racke K. Non-

neuronal acetylcholine, a locally acting

molecule, widely distributed in biological

systems: expression and function in

humans. Pharmacol Ther 1998;77:59–79.

3. Kawashima K, Fujii T. Extraneuronal

cholinergic system in lymphocytes. Phar-

macol Ther 2000;86:29–48.

4. Grando SA, Horton RM. The keratino-

cyte cholinergic system with acetylcholine

as an epidermal cytotransmitter. Curr

Opin Dermatol 1997;4:262–268.

5. Nguyen VT, Hall LL, Gallacher G et al.

Choline acetyltransferase, acetylcholinest-

erase, and nicotinic acetylcholine recep-

tors of human gingival and esophageal

epithelia. J Dent Res 2000;79:939–949.

6. Nguyen VT, Ndoye A, Grando SA. Novel

human a9 acetylcholine receptor regulat-

ing keratinocyte adhesion is targeted by

pemphigus vulgaris autoimmunity. Am J

Pathol 2000;157:1377–1391.

7. Grando SA. Biological functions of kera-

tinocyte cholinergic receptors. J Invest

Dermatol Symp Proc 1997;2:41–48.

8. Grando SA, Crosby AM, Zelickson BD,

Dahl MV. Agarose gel keratinocyte out-

growth system as a model of skin

reepithelization: requirement of endoge-

nous acetylcholine for outgrowth initia-

tion. J Invest Dermatol 1993;101:804–810.

9. Chomczynski P, Sacchi N. Single-step

method of RNA isolation by acid guan-

idinium thiocyanate- phenol-chloroform

extraction. Anal Biochem 1987;162:156–

159.

10. Ndoye A, Buchli R, Greenberg B et al.

Identification and mapping of keratino-

cyte muscarinic acetylcholine receptor

subtypes in human epidermis. J Invest

Dermatol 1998;111:100–106.

11. Nguyen VT, Ndoye A, Hall LL et al.

Programmed cell death of keratinocytes

culminates in apoptotic secretion of a

humectant upon secretagogue action of

acetylcholine. J Cell Sci 2001;114:1189–

1204.

12. Buchli R, Ndoye A, Rodriguez JG, Zia S,

Webber RJ, Grando SA. Human skin

fibroblasts express m2, m4, and m5 sub-

types of muscarinic acetylcholine recep-

tors. J Cell Biochem 1999;74:264–277.

13. Grando SA, Zelickson BD, Kist DA et al.

Keratinocyte muscarinic acetylcholine

receptors: immunolocalization and partial

characterization. J Invest Dermatol 1995;

104:95–100.

14. Andre C, Guillet JG, De Backer JP,

Vanderheyden P, Hoebeke J, Strosberg

AD. Monoclonal antibodies against the

native or denatured forms of muscarinic

acetylcholine receptors. EMBO J

1984;3:17–21.

15. Andre C, Marullo S, Convents A et al. A

human embryonic lung fibroblast with a

high density of muscarinic acetylcholine

receptors.EurJBiochem1988;171:401–407.

16. Carsi-Gabrenas JM, Van der Zee EA,

Luiten PG, Potter LT. Non-selectivity of

the monoclonal antibody M35 for sub-

types of muscarinic acetylcholine recep-

tors. Brain Res Bull 1997;44:25–31.

17. McLeskey SW, Wojcik WJ. Identification

of muscarinic receptor subtypes present in

cerebellar granule cells: Prevention of tri-

tiated propylbenzilylcholine mustard

binding with specific antagonists. Neuro-

pharmacology 1990;29:861–868.

18. Beutner EH, Chorzelski TP, Jablonska S.

Immunofluorescence tests. Clinical signif-

icance of sera and skin in bullous diseases.

Int J Dermatol 1985;24:405–421.

19. Hootman SR, Picado-Leonard TM,

Burnham DB. Muscarinic acetylcholine

receptor structure in acinar cells of mam-

malian exocrine glands. J Biol Chem

1985;260:4186–4194.

20. Bartek J, Staskova Z, Draetta G, Lukas J.

Molecular pathology of the cell cycle in

human cancer cells. Stem Cells

1993;11:51–58.

21. Waseem NH, Lane DP. Monoclonal

antibody analysis of the proliferating cell

nuclear antigen (PCNA). Structural con-

servation and the detection of a nucleolar

form. J Cell Sci 1990;96:121–129.

22. Klapproth H, Reinheimer T, Metzen J

et al. Non-neuronal acetylcholine, a sig-

nalling molecule synthesized by surface

cells of rat and man. Naunyn Schmiede-

bergs Arch Pharmacol 1997;355:515–523.

23. Hebb CO, Krnjevic K. The physiological

significance of acetylcholine. In: Elliott

KAC, Page IH, Quester JH, eds. Neuro-

chemistry, 2nd edn. Springfield: Charles C.

Thomas, 1962: 452–521.

24. Wessler I, Kirkpatrick CJ, Racke K. The

cholinergic �pitfall�: acetylcholine, a uni-

versal cell molecule in biological systems,

including humans. Clin Exp Pharmacol

Physiol 1999;26:198–205.

25. DiPalma JR. Basic Pharmacology in

Medicine, 4th edn. West Chester: Medical

Surveillance Inc, 1994: 880 p.

26. Wang H. Modulation by nicotine on

muscarinic receptor-effector systems.

Chung Kuo Yao Li Hsueh Pao 1997;18:

193–197.

27. Erskine L, McCaig CD. Growth cone

neurotransmitter receptor activation

modulates electric field-guided nerve

growth. Dev Biol 1995;171:330–339.

28. Eberhard DA, Holz RW. Cholinergic

stimulation of inositol phosphate forma-

tion in bovine adrenal chromaffin cells:

distinct nicotinic and muscarinic mecha-

nisms. J Neurochem 1987;49:1634–1643.

88 Arredondo et al.

29. Dinger BG, Almaraz L, Hirano T et al.

Muscarinic receptor localization and

function in rabbit carotid body. Brain Res

1991;562:190–198.

30. Pelto-Huikko M, Dagerlind A, Kononen J

et al. Neuronal regulation of c-fos, c-jun,

and jun-b immediate-early genes in rat

adrenal medulla. J Neurosci 1995;15:1854–

1868.

31. Evinger MJ, Ernsberger P, Regunathan S,

Joh TH, Reis DJ. A single transmitter

regulates gene expression through two

separate mechanisms: Cholinergic regula-

tion of phenylethanolamine N-methyl-

transferase mRNA via nicotinic and

muscarinic pathways. J Neurosci 1994;

14:2106–2116.

32. Falugi C, Pieroni M, Moretti E. Cholin-

ergic molecules and sperm functions.

J Submicrosc Cytol Pathol 1993;25:63–69.

33. Bencherif M, Lukas RJ. Cytochalasin

modulation of nicotinic cholinergic recep-

tor expression and muscarinic receptor

function in human TE671/RD cells a pos-

sible functional role of the cytoskeleton.

J Neurochem 1993;61:852–864.

34. Alkondon M, Pereira EF, Cortes WS,

Maelicke A, Albuquerque EX. Choline is

a selective agonist of a7 nicotinic acety-

lcholine receptors in the rat brain neurons.

Eur J Neurosci 1997;9:2734–2742.

35. Ulus IH, Millington WR, Buyukuysal RL,

Kiran BK. Choline as an agonist: deter-

mination of its agonistic potency on cho-

linergic receptors. Biochem Pharm 1988;

37:2747–2755.

36. Eglen RM, Reddy H, Watson N. Selective

inactivation of muscarinic receptor

subtypes. Int J Biochem 1994;26:1357–

1368.

37. Hulme EC, Birdsall NJ, Buckley NJ.

Muscarinic receptor subtypes. Annu Rev

Pharmacol Toxicol 1990;30:633–673.

38. Caulfield MP. Muscarinic receptors char-

acterization coupling and function. Phar-

macol Ther 1993;58:319–379.

39. Hosey MM. Diversity of structure, sig-

naling and regulation within the family of

muscarinic cholinergic receptors. FASEB

J 1992;6:845–852.

40. Buchli R, Ndoye A, Webber RJ, Grando

SA. Identification and characterization

of muscarinic acetylcholine receptor sub-

types expressed in human skin melano-

cytes. Mol Cell Biochem 2001;228:57–72.

41. MacCallum DE, Hall PA. Biochemical

characterization of pKi67 with the identi-

fication of a mitotic-specific form associ-

ated with hyperphosphorylation and

altered DNA binding. Exp Cell Res

1999;252:186–198.

42. Tsurimoto T. PCNA binding proteins.

Front Biosci 1999;4:849–858.

43. Bartek J, Lukas J. Pathways governing

G1/S transition and their response to

DNA damage. FEBS Lett 2001;490:117–

122.

44. Fujii T, Kawashima K. Ca2+ oscillation

and c-fos gene expression induced via

muscarinic acetylcholine receptor in

human T- and B-cell lines. Arch Phar-

macol 2000;362:14–21.

45. Pinkas-Kramarski R, Stein R, Linden-

boim L, Sokolovsky M. Growth factor-

like effects mediated by muscarinic

receptors in PC12M1 cells. J Neurochem

1992;59:2158–2166.

46. Lindenboim L, Pinkas-Kramarski R,

Sokolovsky M, Stein R. Activation of

muscarinic receptors inhibits apoptosis in

PC12M1 cells. J Neurochem 1995;64:

2491–2499.

47. Yan G-M, Lin S-Z, Irwin RP, Paul SM.

Activation of muscarinic cholinergic

receptors blocks apoptosis of cultured

cerebellar granule neurons. Mol Phar-

macol 1995;47:248–257.

48. Kebabian JW, Neumeyer JL, eds. The RBI

handbook of receptor classification. Natick:

Research Biochemicals International,

1994: p. 122.

49. Zia S, Buchli R, Ndoye A, Greenberg BM,

Nguyen VT, Grando SA. The m4 kerati-

nocyte muscarinic receptor regulates cell

motility and modulates intracellular cal-

cium. J Invest Dermatol 1998;110:545.

50. Williams CL, Lennon VA. Activation of

muscarinic acetylcholine receptors inhibits

cell cycle progression of small cell lung

carcinoma. Cell Regul 1991;2:373–382.

51. Kohn EC, Alessandro R, Probst J,

Jacobs W, Brilley E, Felder CC. Identifi-

cation and molecular characterization of a

m5 muscarinic receptor in A2058 human

melanoma cells: Coupling to inhibition of

adenylyl cyclase and stimulation of

phospholipase A2. J Biol Chem 1996;271:

17476–17484.

Gingival keratinocyte muscarinic receptors 89