©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Regulation of Muscarinic Acetylcholine Receptor Expression during Embryonic Development (*)

(Received for publication, May 3, 1995; and in revised form, June 15, 1995)

Lise A. McKinnon Neil M. Nathanson (§)

From the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7750

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We used solution hybridization, immunoprecipitation, and immunoblot analyses to examine the developmental expression of chicken m2 (cm2), cm3, and cm4 muscarinic acetylcholine receptor (mAChR) mRNA and protein in embryonic and post-hatched chick heart and retina in order to correlate developmental expression patterns with known physiological events. cm2 is the predominant mAChR subtype expressed in chick heart. cm3 and cm4 protein and mRNA expression is very low in chick heart, and cm3 expression is highest early in development. The decrease in cm3 expression correlates well with the developmental decrease in mAChR-mediated activation of phospholipase C. cm4 is the predominant mAChR subtype expressed in chick retina. The expression of both cm4 protein and mRNA is highest early in development and decreases as development progresses. cm2 and cm3 mAChR are expressed at approximately equivalent levels and have similar patterns of expression. The cm2 and cm3 protein levels increase throughout development, while cm2 and cm3 mRNA levels peak at embryonic day 15 and then decrease after hatching. Our data indicate that the three mAChR subtypes are differentially regulated in chick heart and retina and that the patterns of expression of mAChR may be important in the development and physiology of these tissues.


INTRODUCTION

Muscarinic acetylcholine receptors (mAChR) (^1)are a family of neurotransmitter receptors which belong to a superfamily of proteins with seven putative transmembrane domains and elicit their cellular signals through interactions with GTP binding regulatory proteins (G proteins) (reviewed in Nathanson(1987)). Molecular cloning studies have revealed five different subtypes of mammalian mAChR that are products of distinct genes (Bonner et al., 1987, 1988; Hulme et al., 1990). Subtypes m1, m3, and m5 have a relatively high degree of similarity and preferentially couple to the stimulation of phospholipase C, while m2 and m4 are similar and preferentially couple to the inhibition of adenylyl cyclase. Three chicken mAChR subtypes have recently been cloned and designated cm2 (Tietje and Nathanson, 1991), cm3 (Gadbut and Galper, 1994), and cm4 (Tietje et al., 1990). All three subtypes are expressed in chick heart and brain as detected by Northern blot and RNase protection analyses.

Muscarinic receptors undergo several changes in the developing chick heart (reviewed in Nathanson(1989)). Atria from 3-4-day chick embryos display a reduced responsiveness to the negative chronotropic effects of muscarinic agonists even though the density of mAChR is similar to that found in the atria of older embryos (Pappano and Skowronek, 1974; Galper et al., 1977, Renaud et al., 1980). The onset of the negative chronotropic response coincides with structural and functional changes in G(i) that increase mAChR-G(i) coupling (Halvorsen and Nathanson, 1984). The ability of muscarinic agonists to stimulate phosphoinositide turnover is greatest in embryonic day 4 (E4) hearts, and as development progresses this response decreases (Orellana and Brown, 1985; Barnett et al., 1990). There is a developmental increase in the number of mAChRs in chick atria, but not ventricles, between E10 and E12 (Kirby and Aronstam, 1983; Luetje et al., 1987a). This increase in receptor number coincides with the functional innervation of the atria by cholinergic neurons of the parasympathetic nervous system. Blockade with atropine, a muscarinic antagonist (Kirby and Aronstam, 1983) or disruption of innervation (Kirby et al., 1985) can prevent the increase in mAChR number, suggesting that the functional innervation stimulates the developmental increase in atrial mAChR. There is a developmental increase in mAChR number in chick ventricle at the time of hatching (Sullivan et al., 1985; Hosey et al., 1985).

Several studies have shown developmental changes in the density and molecular weight of the mAChR during retinal development in chick embryos. Sugiyama et al.(1977) reported a 30-fold increase in the concentration of mAChR binding sites between E6 and E14, followed by a small decrease in receptor binding sites after hatching. Autoradiographic studies of radiolabeled mAChR showed two bands in the inner plexiform layer in E13 retina and three bands in the inner plexiform layer of adult chicken retina (Sugiyama et al., 1977). SDS-polyacrylamide gel electrophoresis analysis has shown two species of mAChR are present in embryonic chick retina, 86 and 72 kDa (Large et al., 1985); it was not known if these two molecular mass species were derived from distinct genes or posttranslational modifications of a single gene product. The larger form is predominant earlier in development, and after synaptogenesis the smaller form is predominant. It has been suggested that a secreted factor is involved in regulating the switch in expression from the larger to the smaller species (Skorupa and Klein, 1993).

In this study we demonstrate that the expression of cm2, cm3, and cm4 mAChR subtypes and their mRNAs in embryonic chick heart and retina is differentially regulated throughout the development of these tissues. The unique pattern of expression of each mAChR subtype may play a role in the regulation of development of the embryonic chick heart and retina.


MATERIALS AND METHODS

Cloning of the Third Cytoplasmic Loop of the cm3 mAChR

The third cytoplasmic loop of the cm3 mAChR gene was amplified from chicken genomic DNA by the polymerase chain reaction (PCR) with Taq polymerase (Promega) using degenerate oligonucleotides complementary to sequences in the putative fifth and sixth transmembrane domains and the third cytoplasmic loop of the receptor. The following oligonucleotides were synthesized by the University of Washington Molecular Pharmacology Facility. BamHI and EcoRI restriction sites are indicated by brackets: 5` primer 1a: 5`-[GAATTCGGATCC]AC(A/C/T)AT(C/T)ATG(A/T)(G/C)CAT(C/T)CTGTA(C/T)TGG-3`; 5` primer 1b: 5`-[GAATTCGGATCC]AC(A/C/T)AT(C/T)ATGTC(C/T)AT(C/T)CTGTA(C/T)TGG-3`; 5` primer 2a: 5`-[GAATTCGGATCC]TT(C/T)TGGCTGAC(C/A/ T)ATGAA(G/A)(A/T)(G/C)CTGG-3`; 5`primer 2b: 5`-[GAATTCGGATCC]TT(C/T)TGGCTGAC(C/A/T)ATGAA(G/A)TC(C/T)TGG-3`; 3` primer 1: 5`-TGGAC(C/A/T)CC(C/A/T)TA(C/T)AA(C/T)AT(C/T)ATG[GGATCCGAATTC]-3`; 3` primer 2a: 5`-TGGCTGAC(C/A/T)ATGAA(A/G)(A/ T)(G/C)CTGGGA(A/G)[GGATCCGAATTC]-3`; 3` primer 2b: 5`-TGGCTGAC(C/A/T)ATGAA(A/G)TC(C/T)TGGGA(A/G)[GGATCCGAATTC]-3`. Using 5` primer 1a, 5` primer 1b, and 3` primer 1 an 800-base pair fragment was PCR-amplified, isolated, and ligated into the BamHI and EcoRI sites of pGEM3z (Promega), and designated cm3-800. Using cm3-800 as a template, we PCR-amplified a 597-base pair product using 5` primer 2a, 5` primer 2b, and 3` primer 1, and a 234-base pair product using 5`primer 1a, 5` primer 1b, 3` primer 2a, and 3` primer 2b. These PCR products were both ligated into the BamHI and EcoRI sites of pGEM3z. cm3-597 and cm3-234 were then sequenced by the dideoxy chain termination method (Sanger et al., 1977) using Sequenase (U.S. Biochemical Corp.). Sequence analysis indicated that these fragments encoded the third cytoplasmic loop of an m3 subtype of mAChR. Subsequent comparison with the recently published sequence of cm3 (Gadbut and Galper, 1994) confirmed that we had cloned the third cytoplasmic loop of cm3.

RNA Probe Construction

Subtype-selective riboprobes were generated from the third cytoplasmic loop of each mAChR gene. The probes for cm2 and cm4 were described previously (Habecker and Nathanson, 1992). A 363-base pair fragment corresponding to nucleotides 1135-1497 of the coding region of the cm3 gene was PCR-amplified from cm3-597 and ligated into the BamHI and EcoRI sites of pGEM3z. T7 and SP6 RNA polymerases (Promega) were used to transcribe antisense and sense riboprobes, respectively. Labeled riboprobes were separated from unincorporated nucleotides on a Sephadex G-50 RNA spin column (Boehringer Mannheim).

Isolation of Total Nucleic Acids

Total nucleic acids were isolated according to the protocol of McKnight et al. (1988). Tissues were immediately frozen in liquid nitrogen upon dissection and stored at -70 °C. Tissue (100-200 mg) was homogenized in 50 µg/ml proteinase K in 1 SET (1% SDS, 5 mM EDTA, 10 mM Tris, pH 7.5) using a Polytron homogenizer and digested for 1 h at 45 °C. Samples were then phenol/chloroform/isoamyl alcohol-extracted, and nucleic acids were precipitated in 70% EtOH with 150 mM NaCl. Total nucleic acid pellets were resuspended in 0.1 SET and quantitated by UV spectrophotometry. DNA concentration in the total nucleic acid samples was measured by Hoechst stain (Sigma) (Labarca and Paigen, 1980) using a fluorometer.

Solution Hybridization

mAChR mRNA present in 15-20 µg of each of the total nucleic acid samples was quantitated by the solution hybridization assay as described by Habecker and Nathanson (1992). Molecules of specific mAChR mRNA per cell were calculated by comparing samples to a standard curve generated by hybridization of 0.05-10 pg of the sense riboprobes with the antisense riboprobes.

Antibody Production

We designed glutathione S-transferase mAChR fusion proteins to generate subtype-selective mAChR antibodies. PCR was used to amplify a subtype-specific region of the third cytoplasmic loop of each mAChR. Oligonucleotide primers were synthesized by the University of Washington Molecular Pharmacology Facility. The sequence of cm2 that was amplified corresponds to amino acids 253-345. For cm4 the sequence amplified corresponds to amino acids 271-383. These two fragments were each ligated into the BamHI site of the pGEX-2T vector (Pharmacia Biotech Inc.). The sequences amplified for cm3 were amino acids 379-499 and 379-434. These PCR fragments were ligated into the BamHI and EcoRI sites of pGEX-2T. Fusion proteins were purified from bacteria using a glutathione-Sepharose (Pharmacia) column. Antisera were raised in New Zealand White rabbits by R & R Rabbitry (Stanwood, WA). The polyclonal cm2, cm3, and cm4 antisera were affinity-purified using the mAChR fusion proteins coupled to CNBr-Sepharose (Pharmacia). Antibodies were tested for specificity by an immunoprecipitation assay (described below) using membranes from Y1 mouse adrenal carcinoma cells stably expressing either cm2 or cm4 or using membranes from COS-7 green monkey kidney cells transiently expressing cm3 (cm3 in pED was a gift from Dr. Jonas B. Galper).

Cell Culture and Transfection

The Y1 cell lines stably expressing cm2 (Tietje and Nathanson, 1991) or cm4 (Tietje et al., 1990) were grown as described previously. COS-7 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Life Technologies, Inc.), penicillin G (100 units/ml), and streptomycin sulfate (0.1 mg/ml; Apothecon) in a 10% CO(2) environment at 37 °C. COS-7 cells plated on 150-mm culture dishes were transiently transfected with 30 µg of cm3 in pED by the calcium phosphate precipitation method (Sambrook et al., 1989).

Immunoprecipitation Assay

Immunoprecipitation was carried out as described by Luetje et al. (1987b). Tissues were immediately frozen in liquid nitrogen upon dissection and stored at -70 °C. Crude membrane fractions were prepared as described by Luetje et al. (1987b). The mAChR in the membranes were labeled with 2.4 nM [^3H]quinuclidinyl benzilate (47 Ci/mmol; Amersham Corp.) at room temperature and solubilized as described (Luetje et al., 1987b). Solubilized proteins were incubated overnight at 4 °C with goat anti-rabbit Immunobeads (Bio-Rad or Sigma) coated with preimmune or subtype-specific mAChR antibodies. Following incubation, the Immunobeads were pelleted and the supernatant was collected. Radiolabeled mAChR remaining in the supernatant were precipitated with 2 volumes of 75% saturated ammonium sulfate, collected by filtration over GF/C filters, and counted by liquid scintillation. The pelleted Immunobeads were collected by filtration over GF/C filters and counted by liquid scintillation to directly quantitate the percentage of mAChR immunoprecipitated by the Immunobeads coated with the subtype-specific antibodies. The percentage of mAChR precipitated from tissue extracts was normalized by (a) subtraction of the percentage of counts per second immunoprecipitated by Immunobeads coated with preimmune serum and (b) correction for the fraction of receptor immunoprecipitated by each antibody when tested against its respective antigen in transfected cells.

Membrane Preparation for SDS-Gel Electrophoresis

Tissues were homogenized in cold phosphate-buffered saline containing protease inhibitors, and membranes were prepared and stored at -70 °C as described (Luetje et al., 1987a).

Immunoblot Analysis of mAChR Expression

50 µg of membrane protein were solubilized at room temperature for 15 min in SDS-urea sample buffer (3.5% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.0005% bromphenol blue, 8 M urea, and 125 mM Tris-HCl, pH 6.8; prepared fresh using 5 concentrated stock of SDS sample buffer without urea). Proteins were electrophoresed on a 3.5% polyacrylamide stacking gel containing 4 M urea and a 7% polyacrylamide running gel containing 4 M urea (Hunter and Nathanson, 1986) and transferred electrophoretically to Immobilon-P transfer membrane (Millipore Corp.). Immunoblot analysis was performed as described previously (Luetje et al., 1987a). Transfer membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.1% Tween 20) overnight at 4 °C. After washing in TBST, transfer membranes were incubated first with purified anti-cm2, anti-cm3, or anti-cm4 antibody and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Both the primary and secondary antibodies were diluted in 1% bovine serum albumin in TBST and incubated at room temperature for 60 min. ECL Western blotting detection reagents (Amersham Corp.) were used for detection of mAChR immunoreactivity. In some cases, transfer membranes were stripped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris HCl, pH 6.7, at 50 °C for 30 min. and reprobed using the same antibodies; this stripping and reprobing was found to decrease nonspecific labeling.


RESULTS

Specificity of mAChR Antibodies

The efficiency and specificity of the subtype-specific mAChR antibodies was determined by immunoprecipitation of radiolabeled mAChR solubilized from cell lines expressing cm2, cm3, or cm4 (Fig. 1). The affinity-purified cm2 antibody immunoprecipitates 80% of radiolabeled cm2 receptors expressed in stably transfected Y1 cells. Affinity-purified cm4 antibody immunoprecipitates 70% of cm4 receptors from stably transfected Y1 cells, and affinity-purified cm3 antibody immunoprecipitates 79% of cm3 receptors transiently expressed in COS-7 cells. These antibodies show minimal cross-reactivity with the other receptor subtypes, confirming their specificity for their respective antigens.


Figure 1: Immunoprecipitation demonstrating specificity of mAChR antisera . Purified anti-cm2, anti-cm3, and anti-cm4 antibodies were tested for specificity by the immunoprecipitation assay described under ``Materials and Methods.'' Receptors were solubilized from membranes of Y1 cells stably expressing cm2 or cm4 and from membranes of COS-7 cells transiently expressing cm3. Each antibody was used at a final concentration of 6 µg/ml. Data are presented as percentage of cm2, cm3, or cm4 mAChR immunoprecipitated from the total mAChR population in Y1-cm2 cells, Y1-cm4 cells, or COS-7-cm3 cells. Values are mean ± S.D., n = 2-4. Solidbars, cm2; stripedbar, cm3; shadedbars, cm4.



mAChR Protein Expression in Embryonic Chick Heart

The developmental pattern of expression of mAChR proteins in embryonic chick heart was determined by immunoprecipitation to quantitate the percentage of cm2, cm3, and cm4 proteins in the total mAChR population at each stage of development studied. The predominant mAChR protein expressed throughout the development of the embryonic chick heart is cm2 (Fig. 2a). The cm2 protein represents 98-100% of all radiolabeled mAChR in both atria and ventricles at each developmental stage examined. cm3 and cm4 mAChR proteins represent a very small percentage of the total mAChR population in embryonic chick heart, ranging from <1-5% of the total mAChR protein (Fig. 2, b and c). While there seems to be no obvious trend in the developmental expression pattern of cm4 protein, there is a developmental decrease in cm3 protein expression. The level of cm3 in ventricle decreases during development, with statistically significant differences in protein expression between E6V and E9V, E15V, and post-hatched day 7 (P7)V and between E9V and P7V. Although the atria exhibited a trend of decreased expression of cm3 during development, the differences in expression levels are not significant (p > 0.05). These studies are consistent with the observations of Orellana and Brown(1985) and Barnett et al.(1990) that the mAChR-stimulated phosphoinositide turnover is greatest early in development and decreases as development progresses. The cm3 protein is most likely responsible for this phosphoinositide turnover seen in early development.


Figure 2: Immunoprecipitation assay of the expression of cm2, cm3, and cm4 mAChR protein in embryonic and P7 chick heart. Receptors solubilized from membranes of chick atria (A), ventricle (V), or whole heart (H) at various stages of development were used in the immunoprecipitation assay with purified anti-cm2 (a), anti-cm3 (b), and anti-cm4 (c) antibodies. Antibodies were used at a final concentration of 6 µg/ml. The data are presented as percentage of cm2, cm3, or cm4 mAChR immunoprecipitated from the total mAChR population in the chick heart tissues indicated. Values are mean ± S.E., n = 4-10. Statistically significant differences between ventricle samples in panelb are denoted by asterisks (two-tailed t test: *, statistically different from 6V, p < 0.01;**, statistically different from 9V, p < 0.05).



mAChR Protein Expression in Embryonic Chick Retina

We used the immunoprecipitation assay to determine the pattern of expression of cm2, cm3, and cm4 mAChR subtypes during the development of the chick retina. The expression of cm2 protein increases as development progresses, as shown in Fig. 3a. At E9, the cm2 protein is undetectable, and by P7 cm2 represents nearly 50% of total mAChR protein. In contrast, the cm4 protein, which is more highly expressed than cm2 in the retina, decreases as development proceeds. cm4 represents over 70% of mAChR at E9 and decreases to 45% by P7 (Fig. 3c). The pattern of expression of cm3 protein is similar to that seen for cm2: a continual increase as development progresses. At E9 cm3 represents 15% of mAChR protein in the retina and increases to approximately 45% by P7 (Fig. 3b). These data show that there are differential expression patterns among the mAChR proteins expressed in embryonic and post-hatched chick retina. It should be noted that in both the heart and retina, the combined percentage of cm2, cm3, and cm4 mAChR immunoprecipitated was greater than 100%. This is likely due to the normalization in our calculations to correct for the inability of each specific antibody to immunoprecipitate 100% of the respective antigen in the transfected cell lines.


Figure 3: Immunoprecipitation assay of the expression of cm2, cm3, and cm4 mAChR protein in embryonic and P7 chick retina. Receptors solubilized from membranes of chick retina (R) at several stages of development were used in the immunoprecipitation assay with purified anti-cm2 (a), anti-cm3 (b), and anti-cm4 (c) antibodies. Antibodies were used at a final concentration of 6 µg/ml. The data are presented as percentage of cm2, cm3, or cm4 mAChR immunoprecipitated from the total mAChR population in chick retina. Values are mean ± S.E., n = 5-11.



mAChR mRNA Expression in Embryonic Chick Heart

Solution hybridization was used to measure the number of molecules of cm2, cm3, and cm4 mRNA expressed per cell in embryonic chick heart and retina. Consistent with the immunoprecipitation data, the predominant mAChR mRNA in heart is that encoding cm2 (Fig. 4a). The level of cm2 mRNA expression ranged from 5 to 30 molecules/cell. Although the level of cm2 mRNA does not vary greatly during the embryonic stages of development of the atria and ventricle except for an overall decrease after E4, there is an increase in the level of cm2 mRNA in P7 ventricle. In addition, the difference in cm2 mRNA expression between P7 atria and ventricle is substantially greater than any differences between embryonic atria and ventricle. The increase in cm2 mRNA in P7 ventricle is consistent with previous reports of an increase in mAChR binding sites in ventricle after hatching (Sullivan et al., 1985; Hosey et al., 1985).


Figure 4: Solution hybridization assay measuring the expression of cm2, cm3, and cm4 mRNA in embryonic and P7 chick heart. Total nucleic acids were extracted from chick atria (A), ventricle (V), or whole heart (H) at various stages of development and hybridized with subtype-specific riboprobes for cm2 (a), cm3 (b), and cm4 (c) in the solution hybridization assay. The data are presented as the number of molecules of cm2, cm3, or cm4 mRNA expressed per cell in chick heart. Values are mean ± S.E., n = 3-12. Statistically significant differences between atria and ventricle samples in panelb are denoted by asterisks (two-tailed t test: *, statistically different from 6A, p < 0.01;**, statistically different from 9A, p < 0.01).



Like cm2 mRNA expression, cm4 mRNA expression does not vary greatly between atria and ventricle or over the course of embryonic development except for a general decrease after E4 (Fig. 4c). cm4 mRNA was found to be expressed at 1-2.5 molecules/cell, 5-10 times lower than cm2 mRNA expression. The expression of cm3 mRNA was very low, with the mRNA levels being highest early in development. As shown in Fig. 4b, cm3 mRNA is at its highest level at E4 heart and E6 atria, with the difference in cm3 mRNA levels between atria and ventricle at E6 and E9 being significant (p < 0.01). Our results suggest that the mAChR-stimulated inositol phosphate production, which is greatest in E4 heart and decreases as development progresses (Orellana and Brown, 1985; Barnett et al., 1990), is consistent with the pattern of expression of the cm3 receptor shown here.

mAChR mRNA Expression in Embryonic Chick Retina

The overall level of total mAChR mRNA expression in retina is much lower than that seen in heart. The predominant mAChR mRNA subtype in retina is cm4 (Fig. 5c), in agreement with the immunoprecipitation data (Fig. 3). There were 0.5-3.5 molecules of cm4 mRNA per cell, with expression decreasing in the later stages of development. Approximately 0.4-1.2 molecules of cm2 mRNA and 0.1-0.7 molecules of cm3 mRNA are expressed per cell in embryonic chick retina (Fig. 5, a and b). The peak level of expression of both of these mRNAs occurs around E15. Interestingly, synaptogenesis in the retina occurs at the time when all three mAChR mRNAs are at their peak levels of expression, between E13 and E14 (Large et al., 1985).


Figure 5: Solution hybridization assay measuring the expression of cm2, cm3, and cm4 mRNA in embryonic and P7 chick retina. Total nucleic acids were extracted from chick retina (R) at various stages of development and hybridized with subtype-specific riboprobes for cm2 (a), cm3 (b), and cm4 (c) in the solution hybridization assay. The data are presented as the number of molecules of cm2, cm3, or cm4 mRNA expressed per cell in chick retina. Values are mean ± S.E., n = 5-12.



Immunoblot Analysis of mAChR Expression

The subtype-specific mAChR antibodies were used to compare the molecular weight of specific receptor subtypes with the species previously identified in chick retina using affinity-alkylating ligands (Large et al., 1985). As a control the antibodies were tested on immunoblots for specificity against the stably transfected Y1 cell membrane proteins or the transiently transfected COS-7 cell membrane proteins. The anti-cm2, anti-cm3, and anti-cm4 antibodies exhibit a high level of specificity for their corresponding mAChR and show minimal cross-reactivity (Fig. 6). Immunoreactivity to cm2, cm3, and cm4 was all not detectable in skeletal muscle (Fig. 7). Fig. 7a shows that the cm2 protein is highly expressed in E15 ventricle as expected from the previous data. cm2 is not detectable in E9 retina but is found to be expressed in E15 retina and at even higher levels in P7 retina. The approximate molecular mass of cm2 ranges from 64 to 69 kDa. cm3 expression is too low to be detected in E15 ventricle but is expressed in retina with the level increasing as development continues. Its approximate molecular mass is 92 kDa (Fig. 7b). cm4 is also not detectable in E15 ventricle but is present in the retina at a molecular mass ranging from 86 to 95 kDa, with the level of expression decreasing as development continues (Fig. 7c). The differences in molecular weights compared with previous studies of mAChR in the retina (Large et al., 1985) are most likely due to differences in SDS gel electrophoresis protocols. Based on their sizes and patterns of expression we conclude that cm2 corresponds to the lower molecular weight species and cm4 corresponds to the higher molecular weight species detected previously by affinity alkylation (Large et al., 1985).


Figure 6: Immunoblot analysis of expression of mAChR in transfected cell lines. Membranes were prepared from transfected cell lines, and SDS gel electrophoresis was performed as described under ``Materials and Methods.'' Antibodies were used at a final concentration of 1.2 µg/ml. a, anti-cm2; b, anti-cm3; c, anti-cm4. Membrane proteins were loaded in the following order: Y1 cells (lane1), Y1 cells expressing cm2 (lane2), Y1 cells expressing cm4 (lane3), COS-7 cells (lane4), COS-7 cells expressing cm3 (lane5).




Figure 7: Immunoblot analysis of expression of mAChR in chick heart and retina. Membranes were prepared from chick tissues, and SDS gel electrophoresis was performed as described under ``Materials and Methods.'' Antibodies were used at a final concentration of 1.2 µg/ml. a, anti-cm2; b, anti-cm3; c, anti-cm4. Membrane proteins were loaded in the following order: E15 ventricle (lane1), E9 retina (lane2), E15 retina (lane3), P7 retina (lane4), E15 skeletal muscle (lane5).




DISCUSSION

We show here that the expression of cm2, cm3, and cm4 mAChR subtypes is differentially regulated during embryonic development of the chick heart and retina. While the mammalian heart only expresses the m2 receptor (Peralta et al., 1987), chick heart expresses at least three subtypes of mAChR. Both immunoprecipitation and solution hybridization analyses indicate that cm2 is the predominant subtype of mAChR expressed at the protein and mRNA levels. During embryonic development the expression levels of cm2 protein and mRNA do not vary greatly. However, P7 ventricle shows a large increase in cm2 mRNA expression compared with P7 atria or embryonic atria and ventricle. This large increase in cm2 mRNA is likely to be responsible for the increase in mAChR binding sites in ventricle in newly hatched chicks (Sullivan et al., 1985; Hosey et al., 1985).

The level of cm4 expression in embryonic and P7 chick heart is low compared with that of cm2, in agreement with Northern blot analysis by Tietje and Nathanson(1991), which showed that cm2 is more highly expressed than cm4 in embryonic day 18 (E18) chick heart. The pattern of expression of cm4 protein and mRNA is fairly constant in atria and ventricle over the course of development. However, the expression of cm4 may be important functionally during the development of cardiac tissues expressing both cm2 and cm4 because these receptors have different functional sensitivities: cm4 expressed in both CHO cells and Y1 cells is more sensitive to carbachol than cm2 with respect to the inhibition of adenylyl cyclase (Tietje et al., 1990; Tietje and Nathanson, 1991).

The third mAChR subtype expressed in chick heart is cm3. The cm3 mRNA is expressed at higher levels early in development. The expression of cm3 mRNA is greater in atria compared with ventricle, in agreement with studies by Gadbut and Galper(1994), which showed by RNase protection analysis that cm3 expression is higher in E17 atria than in E17 ventricle. The cm3 receptor protein expression is also highest early in development. These data indicate that the mAChR-stimulated phosphoinositide hydrolysis in embryonic chick heart, a response that is highest in E4 hearts and decreases in both atria and ventricle as development progresses (Orellana and Brown, 1985; Barnett et al., 1990), is most likely mediated by the cm3 receptor. Unlike the mammalian cardiac mAChR, both cm2 and cm4 exhibit high affinity for pirenzepine and AF DX-116 (Tietje et al., 1990; Tietje and Nathanson, 1991). The presence of cm3 in chick heart may explain the mAChR-stimulated phosphoinositide hydrolysis in chick heart, which is relatively insensitive to blockade by pirenzepine (Brown et al., 1985), since cm3 expressed in Chinese hamster ovary cells has low affinity for pirenzepine (Gadbut and Galper, 1994). Muscarinic agonists can produce not only inhibitory but also stimulatory effects on the heart. In chick heart, it has been shown that the stimulatory action is mediated by a mAChR with low affinity for pirenzepine (Portas, 1990) and that the developmental dependence of the stimulatory effect correlates with the developmental changes in mAChR-mediated stimulation of phospholipase C activity (Mubagwa et al., 1992). It has been suggested that the stimulatory effect of muscarinic agonists may be a mechanism to prevent excessive suppression of cardiac activity (Pappano, 1991). Because of the presence of high levels of acetylcholine in uninnervated heart early in development (Coraboeuf et al., 1970), the high levels of cm3 early in development may ensure that the heart maintains its contractile activity prior to the onset of parasympathetic innervation.

Large et al.(1985) identified two species of mAChR in embryonic chick retina, with molecular masses of 86 and 72 kDa. The expression of both 86 and 72 kDa increased between E9 and E17, and between E17 and P10 the expression of 86 kDa decreased, while the expression of 72 kDa continued to increase (Skorupa and Klein, 1993). This change in expression was duplicated during culture of retinal cells for 6 days, and conditioned medium from more mature retinal cell cultures caused young retinal cells to switch their expression pattern after only 2 days in culture. It was suggested that a secreted factor from the older retinal cells is involved in regulating the switch in expression from the 86- to the 72-kDa protein (Skorupa and Klein, 1993).

Our studies demonstrate that there are at least three subtypes of mAChR expressed in chick retina. The predominant receptor expressed in retina is cm4. The pattern of expression of both cm4 protein and mRNA is similar to that seen for the 86-kDa protein. The cm4 receptor expression level is greatest early in development and steadily decreases through the P7 stage of development. The level and pattern of expression of the cm2 and cm3 receptors is very similar. Both cm2 and cm3 proteins are expressed at very low levels in the earliest stage of development studied and continually increase as development progresses. This pattern of expression is identical to that seen for the 72-kDa protein.

Immunoblot analysis of mAChR expression in chick retina using subtype-specific antibodies demonstrates that the cm2 protein is approximately 64-69 kDa, cm3 protein is approximately 92 kDa, and cm4 protein is 86-95 kDa. While the differences in the molecular masses found here compared with those reported by Large et al. (1985) are most likely due to differences in electrophoretic conditions, the molecular weight of cm4 is greater than that of cm2, and the immunoprecipitation data for these two proteins exactly correlates with the affinity alkylation studies. We therefore conclude that cm2 corresponds to the lower molecular weight receptor and cm4 to the higher molecular weight receptor previously reported in chick retina. Because of the lack of resolution inherent in counting gel slices of a mixture of affinity-alkylated receptor subtypes, it is likely that the radiolabeled bands representing cm3 and cm4 proteins were not resolved, and therefore a third mAChR was not identified in chick retina in the previous studies.

The cm2 and cm3 mRNA expression patterns in retina are also similar. The level of expression is low early in development, peaks at E15, and then decreases through the P7 stage. Interestingly, all three mAChR mRNAs present in chick retina are most highly expressed at E15, which coincides with synaptogenesis in the retina. The level of all three mRNA subtypes decreases after synaptogenesis is complete. These results correlate with the studies of Sugiyama et al. (1977), who reported an increase in the concentration of mAChR binding sites in chick retina between E6 and E14, followed by a small decrease in receptor binding sites after hatching.

In conclusion, we have demonstrated that the mAChR subtypes expressed in chick heart and retina are differentially regulated during development. The developmental expression patterns of cm2, cm3, and cm4 correlate with physiological and biochemical changes that take place during the development of these tissues. These results suggest both a role for mAChR in the development of the heart and retina and the possibility that there is a unique functional role for each receptor in the developing chick heart and retina.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant HL30630 and Training Grant GM07750. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, Box 357750, University of Washington School of Medicine, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230.

(^1)
The abbreviations used are: mAChR, muscarinic acetylcholine receptor; G protein, GTP-binding regulatory protein; PCR, polymerase chain reaction; E4, E10, E12, etc., embryonic day 4, 10, 12, etc.; P7 and P10, post-hatched days 7 and 10, respectively; A, atria; V, ventricle.


ACKNOWLEDGEMENTS

We thank Dr. Jonas B. Galper for the gift of cm3 in pED.


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