(Received for publication, May 3, 1995; and in revised form, June 15, 1995)
From the
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.
Muscarinic acetylcholine receptors (mAChR) ()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 that increase
mAChR-G
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.
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.
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).
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.
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.
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.
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).
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.