The muscarinic receptor gene expressed in rabbit parietal cells is the m3 subtype

GASTROENTEROLOGY 1992;103:870-875

The Muscarinic Receptor Gene Expressed in Rabbit Parietal Cells Is the m3 Subtype MASAYOSHI

KAJIMURA,

MICHAEL A. REUBEN, and GEORGE SACHS

Center for Ulcer Research and Education, and Departments of Medicine and Physiology, Veterans Administration Hospital and University of California, Los Angeles, California

To investigate the nature of the muscarinic receptors present on parietal cell membranes, binding studies and polymerase chain reaction (PCR) amplification of parietal cell messenger (m) RNA were undertaken. Displacement of N-[3H]methylscopolamine by various muscarinic antagonists showed displacement with a single affinity. The apparent dissociation constant values were as follows: atropine (nonselective), 1.95t-0.28 nmol/L; pirenzepine (M,), 169 + 24 nmol/L; AF-DX 116 (M,), 1542 +- 33 nmol/L; and hexahydrosiladifenidol (M,), 29 t 3.4 nmol/L. These data confirmed the existence of only an M, receptor linked to acid secretion as defined pharmacologically. PCR amplification of parietal cell mRNA with primers designed for detection of all known muscarinic receptor subtypes showed that only m3 fragments were produced from parieta1 cell mRNA, whereas ml and m2 products could be detected in brain or cardiac mRNA. The m3 nature of the PCR product was confirmed by Southern blotting with 32P-labeled human m3 complementary DNA. Hence the two carbachol affinities and the separable cellular responses following muscarinic activation are caused by separate coupling pathways of the M, receptor.

M

uscarinic stimulation of rabbit parietal cell acid secretion can occur directly. With development of additional muscarinic antagonists, studies on acid secretion* have followed the standard pharmacological procedure for muscarinic subtype classification, using relative affinities of various antagonists.“: It has been shown that the receptors on rat parietal cells’ and human3 and porcine4 gastric mucosa could be classified as M, based on high affinity for the MS-selective antagonist hexahydrosiladifenido1 (HHSiD),’ low affinity for the M, antagonist AFDX 116,6 and intermediate affinity for the M, antagonist pirenzepine.7 However, affinity constants can be influenced by assay conditions’ or by a mixed population of receptors.9 * In this paper, the naming of pharmacologically and genetically defined muscarinic receptor subtype follows the recommendation of the Muscarinic Receptor Nomenclature Committee,’ and the pharmacologically and genetically defined subtypes are represented by capital and lower case letters, respectively.

Wadsworth

Other studies have investigated the coupling mechanisms involving changes of intracellular Ca1’-13 and phosphatidylinositol turnover.10*14~15Using the antagonist 4-DAMP, it has been possible to show a dissociation between Ca release from intracellular stores and Ca entry across the plasma membrane of parietal cells. The latter appeared to be associated with changes in acid secretion. Binding studies showed two apparent affinities for carbachol, the high-affinity site related to Ca entry and acid secretion.13 These findings raised the possibility of two different M, receptor subtypes or different coupling of the same M, receptor protein resulting in distinct ligand affinities. Cloning techniques have shown the presence of five muscarinic receptor subtypes, ml to m5.16-*8 These could be related to M, , M,, or M, responses by comparing appropriate antagonist affinities.‘8-20H~~ever, there are no known selective M, or M, antagonists. Further, although exocrine glands have only an M, subtype pharmacologically, both ml and m3 messenger (m) RNA can be detected in some preparations. In the absence of coupling of the M, receptor to a measured function, or with protein levels below threshold in terms of binding, it is difficult to assess the significance of expression of the ml subtype in these tissues. The present study was undertaken to determine by molecular biological means the nature of the muscarinic receptor on the parietal cell, using primers selective for the known subtypes and polymerase chain reaction (PCR) amplification of message in gastric mucosa, gastric glands, and gastric parietal cells. The data showing only the presence of m3 mRNA correlated well with differential binding assays performed with purified parietal cells. Materials and Methods Chemicals Chemicals were obtained from the following sources: collagenase (type B) from Boehringer Mannheim Biochemicals

(Indianapolis,

IN); pronase

from Calbiochem-

0 1992 by the American Gastroenterological 0016-5085/92/$3.00

Association

MLJSCARINIC RECEPTOR SUBTYPE IN PARIETAL CELL

September 1992

icals (San Diego, CA); Nycodenz from Accurate Chemical & Scientific Corp. (Westbury, NY); N-[3H]methylscopolamine (NMS) ([N-methyl-3H]scopolamine methyl chloride, 78.9 Ci/mmol) and Replinase from New England Nuclear (Boston, MA); [32P]dCTP (deoxycytidine 5’-[a3’P]triphosphate, 3000 Ci/mmol) and multiple DNA labeling system from Amersham (Arlington Heights, IL); M-MLV reverse transcriptase (Moloney murine leukemia virus) from Gibco BRL (Grand Island, NY); and RQl DNase, pd(N)G (hexadeoxyribonucleotides), RNasin ribonuclease inhibitor, and dNTP mix (deoxyribonucleotide triphosphates) from Promega (Madison, WI). All other chemicals were of the highest grade available. HHSiD was a kind gift from Prof. G. Lambrecht, Johann Wolfgang Goethe University, Germany. AF-DX 116 and the oligonucleotide primers of five muscarinic receptor subtypes were kind gifts from Allergan Inc. (Irvine, CA). Gastric

Glands and Parietal

Cell Preparation

Gastric glands and parietal cells were obtained from the gastric mucosa of New Zealand white rabbits (1.5-2.5 kg) as described previously.Z1 Briefly, after collagenase and pronase digestion, the parietal cell-enriched fraction was purified by a Nycodenz gradient as described previously.” Parietal cells and/or gastric glands were used for the following experiments.

Binding Studies Purified parietal cells (>75%) were used for the displacement study of [3H]NMS in the presence or absence of muscarinic antagonists as previously described.13 Displacement of [3H]NMS by muscarinic antagonists was performed in the presence of 0.9 nmol/L [3H]NMS, and specific binding of [3H]NMS was defined as the difference in the presence or absence of 10 pmol/L atropine. Binding of [3H]NMS was analyzed by a nonlinear regression analysis (P-fit: Biosoft, Milton, NJ) and fitted to a one or two binding site models as described previously.13

Extraction of RNA and DNase Treatment Total RNA Sample

of

Brain, atrium, and parietal cells or glands were used for the preparation of RNA by a modification of the LiCl/urea method.23 Total RNA was incubated in a solution containing RQl DNase (1 U/10 ug RNA), RNasin ribonuclease inhibitor (4 U/l0 pg RNA), 10 mmol/L MgCl,, and 0.1 mmol/L dithiothreitol for 30 minutes at 37°C. To assess genomic DNA contamination, 5 ug each of total RNA with or without DNase treatment was analyzed on a formaldehyde-agarose geIZ4

PCR To make complementary (c) DNA from RNA, 1 pg of RNA was incubated with 1.22 U hexanucleotides diluted in 10 mmol/L Tris (pH 8) at 65°C for 5 minutes, then

871

kept on ice. The reaction was performed in a total volume of 50 uL with 200 U M-MLV reverse transcriptase, 10 uL 5X reverse transcriptase buffer, 0.5 mmol/L dNTP mixture, 1 U RNasin ribonuclease inhibitor, and 1 nL nuclease-free bovine serum albumin (Promega) for 1 hour at 37°C. The standard condition of PCR was as follows: denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute, and extension at 72’C for 2.5 minutes except for a lo-minute extension in last cycle. PCR was performed for 40 cycles in a total volume of 20 uL containing 5 uL cDNA medium made as above, 1 U Replinase, 0.2 mmol/L dNTP mixture, 10m5 mol/L tetramethylammonium chloride, and 1 umol/ L each of synthesized oligonucleotides corresponding to the putative first extracellular and the third intracellular segments of each human muscarinic receptor. These domains were chosen because of their low homology between receptor subtype.‘“-‘8 Oligonucleotide primers were synthesized by American Synthesis Inc. (Pleasanton, CA), and the sequence positions and sequences were as follows (numbered in the 5’to 3’direction of sense sequence): ml, 33-51 (5’~CAACATCACCGTCCTGGCA-3’) and 837-855 (5’-CATGGAGCCTTCGTCCTCT-3’); m2, 33-51 (5’~CCTGGCTCTTACAAGTCCT3’) and 807-825 (5’-AACACAGTTTTCAGTCACA-3’); m3, 6-24 (5’-CTTGCACAATAACAGTACA-3’) and 947-965 (5’-GGTTTCCAGCTCTTGGTTG-3’); m4,6-24 (5’-CAACTTCACACCTGTCAAT-3) and 743-761(5’-ACGCTCTGCTTCATTAGTG-3’); and m5,13-31 (S-TCTTACCACAATGCAACCA-3) and 746-764 (5’-CGCAAGCAGGATCTGAACA-3’). PCR products were analyzed on 1.4% agarose gels containing 0.5 pg/mL ethidium bromide with TBE buffer [89 mmol/L Tris base, 89 mmol/L boric acid, and 2 mmol/L ethylenediaminetetraacetic acid (EDTA: pH 8.311 at 80 V. DNA Blot Analysis

of PCR Products

Whole human m3 cDNA was donated by Dr. T. I. Banner.” Human whole m3 cDNA [2.74-kilobase (kb) TthIII-Sac1 fragment) was inserted into the SmaI and Sac1 sites of the Okayama-Berg mammalian expression vector pCD-ps (Figure 1). After amplifying the plasmid by conventional methodsz5 the isolated plasmid was digested by EcoRI, Pvu I, Sal I, and Xho I and the fragment between Xho I and EcoRI (2.6 kb), in which m3 specific first extracellular loop was omitted, was used as probe for blot analysis. The probe was radiolabeled with the multiple DNA labeling system. The PCR products, run on a 1.4% agarose gel, were transferred to a nylon membrane (Nytran; Schleicher & Schull, Keene, NH) as described previously.” DNA was bound to the nylon membrane by irradiation with ultraviolet light at 120,000 uJ/cm” for 30 seconds in a Stratalinker (Stratagene, San Diego, CA) apparatus and prehybridazed with the following prehybridization solution; 50% formamide, 5X SSPE (150 mmol/L NaCl, 5 mmol/L NaH,PO,, 1 mmol/L EDTA, pH 7.4), 1X Denhart’s solution (0.02% Ficoll,0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), 0.1% sodium dodecyl sulfate and 0.1 mg/mL salmon sperm DNA at 42°C for 4 hours. Probe was added to incubate overnight at 42°C. Washing was done at high stringency.

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GASTROENTEROLOGY Vol. 103, No. 3

RI 5.91,

0.03 nmol/L;

pCD-PSIHm3

I 1.97

XHO

PCR Amplification SMA

I 3.16’

XHO

1 2.99

Figure 1. Restriction maps of human m3 subtype (Hm3) gene and pCD-ps. Only restriction enzyme sites relevant to suhcloning and isolation are shown. The 2.6-kh EcoRI, Xho I fragment was used to probe Southern blots after PCR products.

Results Binding Studies The binding of various selective muscarinic ligands was characterized by [3H]NMS displacement. This ligand had been shown previously to have a single class of binding sites in purified parietal cells.13 The dissociation constant of [3H]NMS (0.23 +

T e

2

F ._ m : =

fL

mean + SE, n = 5) was used for calculation of each antagonist dissociation constant using the Cheng and Prusoff equation.‘” Displacement of [3H]NMS by pirenzepine, AF-DX 116, HHSiD, and atropine was fitted by a one site displacement model, with a pseudo-Hill plot slope not different from unity (P < 0.05, one-tailed Student’s t test) (Figure 2). The IC,, (50% median inhibiting concentration) values, dissociation constants, and slope factors are presented in Table 1. Also shown is the dissociation constant of 4-DAMP, obtained previously. The value for AF-DX 116 of 1542 nmol/L excludes the presence of a significant level of M, receptor and the value for pirenzepine displacement of 169 nmol/L compared with values of HHSiD of 29 nmol/L and with 4DAMP of 1.7 nmol/L are characteristic of a M, receptor subtype.

100

50

0

We used primers (19 mers) synthesized against regions of low homology in terms of human cDNA sequences for the different muscarinic subtypes, because the gene sequence for rabbit is unknown. The PCR reaction was run on rabbit RNA isolated from brain, atrium, gastric gland, and purified parietal cells to allow for detection of all the muscarinic subtypes. The predicted product size for the ml [823 base pairs (bp)], m2 (793 bp), and m3 (960 bp) was detected in brain RNA and that for m2 in heart atrium. No evidence from PCR reaction was obtained for either m4 or m5 in rabbit brain, contrary to expectation (Figure 3A). In the case of rabbit gastric glands, which contain parietal and peptic cells as well as mucous neck cells, only an m3 receptor subtype was detected by PCR’

Table 1. IC,,

Values and Apparent Dissociation Constants ofAntagonists to Muscarinic Receptor in Rabbit

-

Parietal Cells, Estimated r3H]NMS by Antagonists

-

GO (nmol/L)

1

I,

12

11

10

I

I

I,

9

6

7

6

I

I

I

I

5

4

3

2

-LOG(Antagonist) (M) Figure 2. Displacement of [3H]NMS by antagonists. [3H]NMS binding to parietal cells in the presence of different concentration of atropine (O), pirenzepine (V), AF-DX 116 (+), and HHSiD (A) was measured as described in Materials and Methods. Plotted points showed mean values without standard error ~4% (n = 4-6). IC, values and apparent dissociation constants are summarized in Table 1.Binding of [3H]NMS in the absence of antagonists was taken as 100%.

Atropine Pirenzepine AF-DX 116 HHSiD 4-DAMP

9.63 832 7582 142 8.18

f + f + rt

1.36 120 164 16 0.65

From Displacement

of

Dissociation constant

Slope

(nmol/L)

factor

1.95 169 1542 29 1.66

f + f + +

0.28 24 33 3.4 0.07

0.94 0.93 0.87 0.95 0.85

+ f f f k

0.03 0.06 0.03 0.04 0.03

NOTE. Results are expressed as mean f SE (n = 4-6). Slope factors (n) from pseudo-Hill plot of each displacement curve were calculated by a linear regression analysis using the following equation: Log [([RL,*]/[RL,*]) - l] = n(Log [LJ - Log IC,,), where [LJ, [RL,‘], and [RL,*] are the concentration of the given ligand and the amount of bound [3H]NMS in the absence and presence of the ligand, respectively.

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MUSCARINIC

1992

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12345

1234523

‘go2

4

12345

RECEPTOR

12345

SUBTYPE

IN PARIETAL

CELL

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1 234523

t

603

Figure 3. Muscarinic receptor subtype gene expression in tissues. (A) PCR of ml to m5 subtype were performed as described in Materials and Methods. Reactions were performed with cDNAs from parietal cell, gastric gland, brain, atrium, and pCD-ps including human m3 cDNA. Each lane contains 1 pmol/L of sense and antisense primers of ml (lane l), m2 (lane 2) m3 (Jane 3) m4 (Jane 4), and m5 subtype nucleotide sequences (Jane 5). (B) Blot analysis of PCR products in tissues. PCR products shown in A were transferred to nylon membrane and hybridized with a labeled restriction fragment of human m3 cDNA probe (2.5 X lo6cpm/mL) at 42°C overnight. Membranes were washed at high stringency (0.1XSSPE, 0.1% sodium dodecyl sulfate, 60°C) and exposed for 30 minutes. The positions of size markers are indicated in the figure.

analysis. This finding was extended to purified parieta1 cells, correlating with the pharmacology discussed above. Southern

Blot Analysis of PCR Product

Because the gene sequence of rabbit muscarinic receptors is unknown, it was important to confirm by an independent method that the PCR product of predicted molecular weight indeed contained the m3 sequence. Blotting with a labeled 2.6-kb fragment of human m3 cDNA, downstream from the primer sequence under stringent wash conditions, gave a signal for short exposure only in the m3 PCR lane for brain, gland, and parietal cell (Figure 3B). Longer exposure (4 hours) showed the presence of signal in the ml lane for brain, due to significant homology between these receptors. Overnight exposure did not show any binding to the ml lane of gastric PCR product, and no m4 or m5 hybridization was observed. These data confirm that the major muscarinic subtype in the secretory gastric epithelium is the m3 subtype. Discussion The data presented above analyzing the pharmacology of the muscarinic system in rabbit glands and parietal cells are consistent with data from this and other laboratories concluding that evidence could only be obtained for a functional M, receptor subtype. The dissociation constant for pirenzepine of 169 nmol/L was similar to that of 189 nmol/L obtained by Pfeiffer et alI5 as was the 29 nmol/L for

HHSiD compared with 6.2 nmol/L in rat parietal cells.’ The values presented in the present study differ slightly from values for rabbit gastric mucosal cells,27 but the order of apparent affinity shows consistency with previous publications. Other exocrine glands seem to differ somewhat in their subtype expression. For example, rat lacrimal and salivary glands seem to contain ml and m3 subtypes,‘* but only m3 (HM4 in the paper) has been detected in rat pancreas.” In the case of the parietal cell we have been able to show only the presence of the M, subtype, because the pharmacology described in the present study is identical to that found for selectively transfected mammalian cells’*~20 or injected Xenopus oocytes.‘g Recently, Waelbrock et al.,” Lazareno et al.,3o and Dorje et a131 reported that M, selective antagonists, himbacine and methoctramine, can discriminate between M, and M,, showing that these antagonists have higher affinity for M, than M,. The dissociation constants of above antagonists in this study are well consistent with pancreas M,, not striatum M,” and these relative affinities are comparable with M, in submandibular glands3’ In addition, M, receptors inhibit cyclic adenosine monophosphate production and there is no evidence for cholinergic inhibition of histamine stimulation of acid secretion in rabbit parieta1 cells, only potentiation3’ However, pharmacological approach cannot exclude the presence of M,, because selective ligand for M, subtype is unknown.’ In fact, it has been shown that the expression of M, receptors in cell lines produces effects identical to those of M, receptors.33 Hence the conclusion from

874

functional and binding studies is that stimulation of acid secretion is caused by the presence of either M, or M, subtype receptors on the parietal cell surface. Under standard PCR conditions we were not successful in detecting either m4 or m5 transcripts in brain RNA, although these have been described based on Northern”*” or on in situ hybridization.34.35 A weak band of the m4 size was found below 54% annealing temperature, but this was not the case for gastric RNA product. The reason for our inability to detect these messages is probably not caused by poor extraction because m2, a low level message in brain,18’34 was readily detectable. Probably the homology between human and rabbit sequence is too poor for the PCR reaction to work satisfactorily for these messages, especially for the m5 subtype for which no message could be detected even at low annealing temperatures. The m3 subtype was readily detected in gastric glands and in purified parietal cells, in the absence of any other detectable PCR product. It would not be expected that m4 or m5 subtypes are present in significant quantities, because they are mainly expressed in brain,‘8’Z8 except for m4 found in rabbit lung.30 In fact, the m5 subtype has not been found in peripheral tissues.17 The identity of the product of the m3 oligomer primed reaction was confirmed to be m3 by Southern blot analysis using a 2.6-kb fragment of human m3 cDNA. It seems, therefore, that the PCR reaction confirms that the M, pharmacological response is a result of expression of m3 message in the secretory cells of the gastric mucosa, with no other receptor subtype message being present at detectable levels. Peptic cells, which are an equal constituent of gastric glands, also only express m3 RNA according to our data. The functional response of gastric glands and parieta1 cells to carbachol stimulation are absolutely dependent on external Ca, showing the necessity of Ca entry for effective stimulation of acid secretion to be observed. These data are obtained in the presence of H, blockers, eliminating a role for histamine release in this muscarinic response. Parietal cells respond to

Figure 4. A model illustrating differential coupling of the M, receptor to Ca’+ entry and to PLC by different G protein (GPI, (2.

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carbachol not only with a steady state increase in intracellular Ca as a result of Ca entry but also with a intracellular Ca transient.“-l3 It has made possible to show that the transient is blocked by low concentrations of the antagonist 4-DAMP, without measurable effect on acid secretion or its related metabolism.13 It was also possible to show high and low affinity sites for displacement of [3H]NMS by carbachol. These data, given the presence of only a m3 subtype muscarinic receptor, must be interpreted on the basis of a pharmacological M, receptor subtype and not because of contamination with any other muscarinic receptor subtype. It is known that the affinity of muscarinic receptor for agonists is influenced by receptor G-protein complex. Indeed, in cell lines individually transfected with ml through m4 subtypes, high and low affinity states for agonists could be detected.” Multiple Gprotein interactions have been described for muscarinic receptors. An m2 receptor purified from baculovirus-transfected insect Sf 9 cells allows binding of guanosine-5’-0-(3-thiotriphosphate) to four kinds of G protein (Gil, G,, G,, and G,), suggesting a natural tendency for multiple G-protein association.36 The combination of functional and receptor identification data suggests that the M3(= m3) receptor in the parietal cell either exists in two affinity states coupled to the same G protein or is coupled to distinct G proteins. The ability to clearly dissociate the functional response and Ca entry from the Ca intracellular transient suggests that the muscarinic phospholipase C/phosphatidylinositol 4,5_biphosphate pathway shown in the parietal ce11’“*‘4is coupled to release of Ca2+ but not to Ca entry and secretion and that a high and low affinity state of the same M,-Gprotein complex does not explain the data. Rather the data suggest that a separate M,-G-protein complex is linked directly to Ca entry and is responsible for the secretory response of the parietal cell (Figure 4). References Birdsall N, Buckley N, Doods H, Fukuda F, Giachetti A, Hammer R, Kilbinger H, Lambrecht G, Mutschler E, Nathanson N, North A, Schwarz R. Nomenclature for muscarinic receptor subtypes recommended by symposium. Trends Pharmacol Sci 1989;1O(Suppl 14). Pfeiffer A, Rochlitz H, Noelke B, Tacke R, Moser U, Mutschler E, Lambrecht G. Muscarinic receptors mediating acid secretion in isolated rat gastric parietal cells are of M3 type. Gastroenterology 1990;98:218-222. Pfeiffer A, Hanack C, Kopp R, Tacke R, Moser U, Mutscbler E, Lambrecht G, Herawi M. Human gastric mucosa expresses glandular M3 subtype of muscarinic receptors. Dig Dis Sci 1990;35:1468-1472. Herawi M, Lambrecht G, Mutschler E, Moser U, Pfeiffer A. Different binding properties of muscarinic M2-receptor sub-

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type for agonists and antagonists in porcine gastric smooth muscle and mucosa. Gastroenterology 1988;94:630-637. Lambrecht G, Moser U, Wagner M, Wess J, Gmelin G, Raseiner K, Strohmann C, Tacke R, Mutschler E. Pharmacological and electrophysiological evidence for muscarinic Ml and M2 receptor heterogeneity. Trends in Pharmacol Sci 1988;(Suppl):82. Hammer R, Giraldo E, Schiavi GB, Manferini E, Ladinsky H. Binding properties of a novel cardioselective muscarinic antagonist, AF-DX 116, to membrane of peripheral tissues and brain in the rat. Life Sci 1986;38:1653-1662. Hammer R, Giachetti A. Muscarinic receptor subtypes: Ml and M2 biochemical and functional characterization. Life Sci 1982;31:2991-2998. Hulme EC, Birdsall NJM, Buckley NJ. Muscarinic receptor subtypes. Annu Rev Pharmacol Toxic01 1990;30:633-673. Watson SP, James W. PCR and the cloning of receptor subtype genes. Trends in Pharmacol Sci 1989;10:346-348. Chew CS, Brown MR. Release of intracellular Ca’+ and elevation of inositol triphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa. Biochem Biophys Acta 1986;888:116-125. Muallem S, Sachs G. Ca*+ metabolism during cholinergic stimulation of acid secretion. Am J Physiol 1985;248:G218-G228. Negulescu PA, Reenstra WW, Machen TE. Intracellular Ca requirements for stimulus-secretion coupling in parietal cell. Am J Physiol 1989;256:C241-C251. Wilkes JM, Kajimura M, Scott DR, Hersey SJ, Sachs G. Muscarinic responses of gastric parietal cells. J Membr Biol 1991;122:97-110. Chiba T, Fisher SK, Park J, Seguin EB, Agranoff BW, Yamada T. Carbamoylcholine and gastrin induce inositol lipid turnover in canine gastric parietal cells. Am J Physiol 1988;255:G99-G105. Pfeiffer A, Rochlitz H, Herz A, Paumgartner G. Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M2 receptor. Am J Physiol 1988;254:G622G629. Bonner TI, Buckley NJ, Young AC, Brann MR. Identification of a family of muscarinic acetylcholine receptor genes. Science 1987;237:527-532. Bonner TI, Young AC, Brann MR, Buckley NJ. Cloning and expression of human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1988;1:403-410. Peralta EG, Ashikenazi A, Winslow JW, Smith DH, Ramachandran J, Capon DJ. Distinct primary structures, ligandbinding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J 1987; 6:3923-3929. Akiba I, Kubo T, Maeda A, Bujo H, Nakai J, Mishina M, Numa S. Primary structure of porcine muscarinic acetylcholine receptor III and antagonist binding studies. FEBS Lett 1988;235:257-261. Buckley NJ, Bonner TI, Buckley CM, Brann MR. Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-Kl cells. Mol Pharmacol 1989;35:469-476. Chew CS, Hersey SJ, Sachs G, Berglindh T. Histamine responsiveness of isolated gastric glands. Am J Physiol 1980; 238:G312-G320. Muallem S, Burnham C, Blissard D, Berglindh T, Sachs G. Electrolyte transport across the basal lateral membrane of the parietal cell. J Biol Chem 1985;260:6641-6653.

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23. Auffray C, Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem 1980;107:303-314. 24. Goldbergs DA. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Nat1 Acad Sci USA 1982;77:5794-5798. 25. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. 26. Cheng YC, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I& of an enzymatic reaction. Biochem Pharmacol 1973;22:3099-3108. 27. Baudiere B, Monferini E, Giraldo E, Ladinsky H, Bali JP. Characterization of the muscarinic receptor subtype in isolated gastric fundic cells of the rabbit. Biochem Pharmacol 1987;36:2957-2961, 28. Maeda A, Kubo T, Mishina M, and Numa S. Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes. FEBS Lett 1988;239:339-342. 29. Waelbrock M, Tastenoy M, Camus J, Christophe J. Binding of selective antagonists to four muscarinic receptors [Ml to M4) in rat forebrain. Mol Pharmacol 1990;38:267-273. 30. Lazareno S, Buckley NJ, Roberts FF. Characterization of muscarinic M4 binding sites in rabbit lung, chicken heart, and NG108-15 cells. Mol Pharmacol 1990;38:805-815, 31. Dorje F, Wess J, Lambrecht G, Take R, Mutschler E, Brann MR. Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Ther 1991;256:727733. 32. Berglindh T. Potentiation by Carbachol and Aminophylline of Histamine and db-CAMP-induced parietal cell activity in isolated gastric glands. Acta Physiol Stand 1977;99:75-84. 33. Liao CF, Schilling WP, Birnbaumer M, Birnbaumer L. Cellular responses to stimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells. J Biol Chem 1990;265: 11273-11284. 34. Buckley NJ, Bonner TI, Brann MR. Localization of a family of muscarinic receptor mRNAs in rat brain, J Neurosci 1988;8:4646-4652. 35. Weiner DM, Levey AL, Brann MR. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia. Proc Nat1 Acad Sci USA 1990;87:7050-7054, 36. Parker EM, Kameyama K, Higashigima T, Ross EM. Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells. J Biol Chem 1991;266:519-527.

Received October 25, 1991. Accepted March 17, 1992. Address requests for reprints to: Masayoshi Kajimura, M.D., Membrane Biology Laboratory, Building 113, Room 324, Center for Ulcer Research and Education, Veterans Administration Medical Center, Wadsworth, Los Angeles, California, 90073. Supported in part by Veterans Administration Medical Research Funds and National Institutes of Health Grant DK 40615 and DK 41301. Dr. Kajimura was a Marion Research Fellow. The authors thank Dr. T. I. Bonner for donating human m3 cDNA and Dr. G. Lambrecht for the gift of haxahydrosiladifenidol. The authors also thank Krister Bamberg for helpful technical assistance and David Scott for help in preparing figures for publication