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(Circulation Research. 1996;79:86-93.)
© 1996 American Heart Association, Inc.

Articles

Molecular and Functional Identification of m₁ Muscarinic Acetylcholine Receptors in Rat Ventricular Myocytes

Virendra K. Sharma, Henry M. Colecraft, David X. Wang, Allan I. Levey, Elena V. Grigorenko, Hermes H. Yeh, Shey-Shing Sheu

the Department of Pharmacology and Physiology (V.K.S., H.M.C., D.X.W., S.-S.S.), School of Medicine and Dentistry, University of Rochester (NY); the Department of Neurology (A.I.L.), Emory University School of Medicine, Atlanta, Ga; and the Department of Physiology and Pharmacology (E.V.G., H.H.Y.), Bowman-Gray School of Medicine, Winston-Salem, NC.

Correspondence to Dr Shey-Shing Sheu, Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642. E-mail sheus@pharmacol.rochester.edu.

	Abstract

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The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m₁ and m₂ but not for the m₃ to m₅ mAChRs. The identity of the m₁ and m₂ bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m₁ and m₂, but not m₃, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m₁ mAChR was responsible for the stimulatory effects on Ca²⁺ transients by high concentrations of carbachol (>10 µmol/L) known to occur in these cells. In pertussis toxin–treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 µmol/L) increased the basal Ca²⁺ level from 96±7 to 118±8 nmol/L and the peak Ca²⁺ transient level from 519±32 to 640±36 nmol/L (mean±SEM, P<.05 for both, n=8). These effects of carbachol on Ca²⁺ transients were antagonized by 10 nmol/L pirenzepine, an m₁ mAChR–selective antagonist. In contrast, the m₂ mAChR–selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m₁ mAChRs are expressed on adult rat ventricular myocytes in addition to m₂ mAChRs. The results further suggest that m₁ mAChRs mediate the stimulatory responses on Ca²⁺ transients to high concentrations of cholinergic agonists seen in these cells.

Key Words: muscarinic acetylcholine receptors • rat heart myocytes • Ca²⁺ transients • carbachol

	Introduction

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Muscarinic acetylcholine receptors mediate a variety of cellular responses, including inhibition of adenylate cyclase, modulation of K⁺ channels, and increased phosphoinositide breakdown.¹ These diverse effects of mAChR activation elicit both negative and positive inotropic and chronotropic effects in the heart.^{2 3 4} The positive inotropic and chronotropic effects of cholinergic agonists are observed only at a high concentration (>10 µmol/L) and are PTX insensitive.⁵ This is in contrast to the negative inotropic effects^{3 4} that are observed at lower concentrations and are sensitive to inactivation by PTX. These dual effects of mAChR activation in heart may be due to the presence of multiple subtypes of mAChRs. So far, five mAChR subtypes (m₁ to m₅) have been identified, each subtype being encoded by a different gene.^{6 7 8 9} Classification of the mAChR subtypes present in heart is still very controversial. A major reason for this is that the pharmacological agents available at present are inadequate to selectively distinguish one subtype in the presence of the others. Thus, binding studies with selective antagonists have reported that neonatal rat cardiomyocytes express only the M₂ mAChR¹⁰ or the M₂ and M₃ mAChRs.¹¹ Furthermore, it has been reported that mAChRs in adult rat heart are mainly composed of the M₂ subtype, with a small percentage of the M₁ subtype.¹² Experimental evidence from another receptor-binding study supported the existence of two subtypes of cardiac M₂ receptors, M₂ and M_2ß, each with two interconvertible states.¹³ This conclusion was made on the basis of agonist binding affinities. These authors also reported that stimulation of M₂ inhibits adenylate cyclase, whereas M_2ß increases phosphoinositide hydrolysis.¹⁴ Conversely, it has been reported that the increase in phosphoinositide accumulation by cholinergic agonists in cardiac cells from newborn rats and guinea pigs is mediated by mAChRs that are neither M₁ nor M₂.¹⁵ Recent pharmacological studies have shown that the excitatory muscarinic response in cultured neonatal rat ventricular myocytes is mediated by PTX-sensitive M₁ mAChRs.¹⁶ The above-mentioned reports, which implicated the presence of multiple mAChR subtypes in cardiac cells, were at variance with molecular studies in which Northern blot analysis yielded evidence supporting the existence of only m₂ mAChRs in cardiac atrium.^{17 18} Recently, however, it has been reported that m₁ mAChR mRNA can be detected in adult guinea pig ventricular cells using RT-PCR.¹⁹ In contrast, only m₂ mAChR mRNA was detected by these authors using Northern blot analysis. These results suggest that the Northern blot technique is not sensitive enough to detect mAChR subtypes expressed at a low level in heart. In the present study, we have used RT-PCR and immunofluorescence with subtype-specific antibodies to probe the expression of mAChR subtypes in rat ventricular myocytes. Initially RT-PCR was performed on total RNA obtained from 2 to 3 million freshly isolated rat ventricular myocytes. However, this preparation, though purified, may be contaminated with other types of cells, such as endothelial cells, fibroblasts, and smooth muscle cells, or nerve endings. To exclude the possibility of amplifying cDNA from noncardiomyocytes, we repeated RT-PCR in freshly isolated individual myocytes (single-cell RT-PCR). We have further investigated whether the dual effects of mAChR activation on the force of contraction are mediated through different receptor subtypes. The results of the present study indicate that PTX-insensitive m₁ mAChRs are present in rat heart ventricular cells and that activation of these receptors by carbachol produces an increase in the magnitude of cytosolic Ca²⁺ transients.

	Materials and Methods

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Cell Isolation and Purification
Heart cells were enzymatically dissociated under sterile conditions from the ventricles of adult male Sprague-Dawley rats (250 to 300 g) by a method described previously.²⁰ Briefly, the isolated heart was perfused with Ca²⁺-free Joklik's tissue culture medium (GIBCO) for 5 minutes to cleanse the heart of blood. The perfusion solution was changed to Joklik's medium containing 50 µmol/L CaCl₂, 0.5 mg/mL collagenase (Worthington, type II), and 0.1% BSA (Sigma Chemical Co). This enzyme solution was recirculated through the heart for 30 minutes, at which time the heart was removed from the perfusion system. The ventricles were shaken vigorously and filtered through 20-µm nylon mesh to dissociate the muscle into single cells. The left and right ventricles were not separated. The dissociated cells used for RNA preparation were purified with Percoll (Pharmacia Fine Chemicals) as described previously.²¹ The cells were suspended in isotonic Percoll diluted with Joklik's tissue culture medium and centrifuged for 5 minutes at 34g. Intact rectangular cells were collected from the bottom of the tube. Each heart yielded 2 to 3 million rod-shaped myocytes.

RNA Preparation
Total RNA was extracted from purified ventricular myocyte suspensions using the Micro-Scale total RNA preparation kit (Clontech). Two million cells were transferred to a microcentrifuge tube containing 1 mL of denaturing solution (4 mol/L guanidinium thiocyanate, 25 mmol/L sodium citrate, 0.5% sodium N-lauroylsarcosine, and 0.1 mol/L 2-mercaptoethanol). The lysed cell suspension was passed through a 26- to 30-gauge needle twice to shear the DNA. This was followed by extraction with phenol and isopropanol precipitation. The RNA samples were purified by centrifugation in 5.7 mol/L cesium chloride solution for 24 hours at 20°C to remove cellular DNA.²² The residual genomic DNA was removed by treating the RNA samples with 1 U RNase-free DNase (Promega) at 37°C for 30 minutes. The quality and integrity of RNA were assessed by the 260/280 optical density readings (ratio of values measured at 260 and 280 nm) and by running a denaturing formaldehyde agarose gel. RNA samples that exhibited a 260/280 ratio of >1.8 were used for RT-PCR.

Reverse Transcription
First-strand cDNA was synthesized using the RT system kit (Promega). The reaction mixture (final volume, 40 µL) contained 1 µg RNA, 5 mmol/L MgCl₂, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1 mmol/L each of dATP, dCTP, dGTP, and dTTP, 1 U/µL RNase inhibitor, 1.0 µmol/L oligo(dT)15 primer, and 2.5 U/µL AMV RT. To ensure the absence of genomic DNA in the RT-PCR reaction, control tubes were also run that contained no RT (negative control). The synthesis of single-stranded cDNA was carried out for 1 hour at 42°C. The reaction was terminated by incubating the reaction mixture at 95°C for 5 minutes.

PCR
Specific primers were designed to direct synthesis of the five known subtypes of mAChRs for the rat. Primer design was optimized using the OLIGO computer program (National Biosciences), and primers of 18 to 21 nucleotides in length were synthesized with an automated DNA synthesizer (Applied Biosystems). Primer sequences, along with positions of the termini of each primer corresponding to numbering of the original published sequence,^{6 7 8 9 23} are indicated as follows: m₁, 5'-GGGAGCTGGCCGCCCTGC-3' (801 to 818) and 5'-GCCTTTCTTGGTGGGCCTC-3' (1141 to 1123) (341); m₂, 5'-TACCCTCTACACTGTGATTGGC-3' (689 to 711) and 5'-ATGATGACAGGCAGATAG-3' (1052 to 1035) (364); m₃, 5'-GTGGTGTGATTGGTCTG-3' (591 to 610) and 5'-TCTGCCGAGGAGTTGGTGTC-3' (1380 to 1361) (790); m₄, 5'-AGTGCTTCATCCAGTTCTTGTCCA-3' (543 to 566) and 5'-CACATTCATTGCCTGTCTGCTTTG-3' (1052 to 1029) (510); and m₅, 5'-CTCATCATTGGCATCTTCTCCA-3' (1199 to 1220) and 5'-GGTCCTTGGTTCGCTTCTCTGT-3' (1649 to 1628) (451).

PCR amplifications were carried out at a final volume of 100 µL buffer containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 2.5 mmol/L MgCl₂, 200 µmol/L of each of the dNTPs, 1 µmol/L of each primer, 2.5 U Ampli Taq DNA polymerase, and 5 µL of the experimental template. The reaction tubes were overlaid with 60 µL of mineral oil to prevent evaporation, and PCR was performed in an automated thermal cycler (Perkin Elmer 480). For the initial denaturation step, the reaction mixture was heated to 95°C for 3 minutes. Thirty cycles (94°C for 1 minute, denaturation; 60°C for 1 minute, annealing; and 72°C for 1 minute, amplification) were carried out, followed by a final extension at 72°C for 7 minutes. Reactions were terminated with stop buffer, and 15 µL of the PCR products were electrophoresed on 1.2% agarose gels containing 0.5 µg/mL ethidium bromide and photographed under fluorescent UV illumination.

Single-Cell RT-PCR
Synthesis of first-strand cDNA and PCR of freshly isolated ventricular myocytes were obtained by following a previously published protocol for acutely dissociated rat retinal cells.²⁴ Briefly, an individual myocyte was first identified by morphology and then harvested by using negative pressure through a glass pipette (2- to 3-µm tip diameter). The entry of the cell membrane and cellular contents into the glass pipette was monitored visually, with care taken to avoid picking up other cells. The content of each pipette was transferred into a sterile Eppendorf tube, which contained a reverse-transcription master mix without RT (final volume, 20 µL). The mixture contained 2 µL 10x RT buffer (500 mmol/L Tris-HCl [pH 8.15 at 42°C], 60 mmol/L MgCl₂, 400 mmol/L KCl, and 10 mmol/L dithiothreitol), 0.5 µmol/L random hexamer primers (Boehringer Mannheim Biochemicals), 0.5 mmol/L of each of the dNTPs (Promega), and 20 U RNase inhibitor (Promega). Genomic DNA was removed by adding 1 U RNase-free DNase (Promega), followed by incubation for 30 minutes at 37°C. The reaction was stopped, and the DNase was inactivated by heating the sample for 5 minutes at 95°C. After cooling of the sample to room temperature, 25 U of RT (Seikagaku America) was added, and the synthesis of single-stranded cDNA was allowed to proceed for 1 hour at 42°C. The negative controls were run as described above, but water was added in place of RT. The PCR reactions were performed as described above for cell suspensions, but the cycle number was increased from 30 to 40.

Restriction Digest Mapping
Products from six PCR samples were combined to obtain sufficient material for restriction digest mapping. The DNA was purified using a SpinBind PCR purification system kit (FMC). The DNA pellets were resuspended in sterile water. The unique restriction sites and the restriction enzymes were selected using the DNA Strider computer program. Restriction enzymes Ava I, Sau3AI, and Pvu II were used for the m₁ fragment; NspBII, Apa I, and Ban II were used for digestion of the m₂-amplified PCR product. The restriction digestion reaction was carried out for 2 hours at 37°C. The digested products were analyzed by electrophoresis on a 5% nondenaturing polyacrylamide gel.

Southern Blot Analysis
Southern blot analysis was performed as described previously.²² Briefly, the PCR products were separated on a 1.5% agarose gel and transferred to BA-S–supported nitrocellulose membrane (Schleicher and Schuell). Prehybridization was performed at 68°C for 5 hours in prehybridization solution containing 6x SSC, 0.5% SDS, 5x Denhardt's reagent, and 100 µg/mL denatured salmon sperm DNA. The DNA probe was synthesized using a random-primer DNA labeling system (Life Technologies). A 1.8-kb DNA fragment coding for mouse m₁ muscarinic receptor protein (a gift from Dr N. Nathanson, University of Washington) was used as the template. Radioactive probe (5x10⁶ cpm/10 mL) was added in the prehybridization solution, and the blot was incubated at 68°C overnight. The membrane blot was then washed twice in 2x SSC and 0.1% SDS at room temperature and twice in 0.1x SSC and 0.5% SDS at 68°C for 1 hour. Membranes were then exposed to Kodak X-OMAT AR X-film for autoradiography for 48 hours at -70°C.

Immunofluorescence Studies
Isolated myocytes were fixed in suspension with 1% paraformaldehyde for 15 minutes. The cells were subsequently permeabilized with PBS containing 0.3% Triton X-100. Nonspecific binding sites were blocked using GPBS. Cells were then exposed to affinity-purified rabbit polyclonal antibodies specific for the m₁ to m₃ mAChRs,²⁵ diluted 1:200 (final concentration, 0.5 µg/mL) in GPBS for 48 hours at 4°C. These antibodies have been extensively characterized by immunoprecipitation, immunoblotting, and immunocytochemistry using the family of cloned mAChRs and the receptors in the native tissues.^{25 26} Excess primary antibody was removed by repeated washing, and the cells were exposed to a fluorescein-labeled goat anti-rabbit secondary antibody (GIBCO), used at 1:200 dilution. After incubating for 45 minutes at room temperature, excess secondary antibody was removed by repeated washing in GPBS. The labeled cells were then mounted on glass slides in Mowiol 4-88 (Calbiochem). Digital imaging was performed on the stage of a Nikon inverted microscope equipped for epifluorescence with a 40x oil-immersion microscope objective. Details of the digital imaging have been published previously.²⁷ The selected cell was excited at 490 nm, and the emitted fluorescence images were acquired at 530 nm with a dichroic mirror focused onto an SIT camera.

Ca²⁺ Transient Recordings
The detailed procedure for recording of Ca²⁺ transients in adult ventricular myocytes has been described in our previous publications.^{28 29} Isolated myocytes were loaded for 15 minutes at room temperature with 1 µmol/L of the fluorescent Ca²⁺ indicator fura 2-AM (Molecular Probes) in HEPES-buffered solution containing (mmol/L) KCl 5, NaCl 140, CaCl₂ 2, MgCl₂ 1, glucose 10, and HEPES 10 (pH 7.4). The coverslip with dye-loaded cells was mounted in a tissue chamber (Bellco) on the stage of a Nikon Diaphot inverted microscope equipped for epifluorescence (Deltascan 1, Photon Technology International). The myocytes were electrically stimulated at a frequency of 1 Hz with an S8 Grass stimulator. The cell was sequentially stimulated at 340- and 380-nm wavelength light using two excitation monochromators at a switching frequency of 100 Hz controlled by an optical chopper. The data collection rate at this switching frequency of 100 Hz was sufficient to resolve the rising and falling phases of the Ca²⁺ transient.

Determination of [Ca²⁺]_i
In vitro calibration was performed to calibrate fluorescence signals. The calibration solution contained 140 mmol/L KCl, 10 mmol/L NaCl, 1 mmol/L K₂ EGTA, 1 mmol/L MgCl₂, 10 mmol/L HEPES, and 3 µmol/L fura 2 pentapotassium salt (adjusted to pH 7.2). Only two calibrating solutions, Ca²⁺ free and saturating Ca²⁺, were needed to determine the entire calibration curve. [Ca²⁺]_i values were calculated from the 340- to 380-nm ratios using the following equation³⁰ :

(E1)

where R is the measured cellular ratio, and R_min and R_max are the ratios obtained in Ca²⁺-free and saturating Ca²⁺ solutions, respectively. Sf₂ is the 380-nm excitation signal in the absence of Ca²⁺, and Sb₂ is the 380-nm excitation signal at saturating Ca²⁺ in calibrating buffer. K_d is the dissociation constant of fura 2 for Ca²⁺.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of mAChR Subtypes in Rat Heart
The effectiveness of the m₁ to m₅ mAChR subtype–specific primers was checked by amplifying all five mAChR sequences from mouse brain.²³ The primers were able to amplify m₁ to m₅ cDNAs, and each PCR product was of the predicted size; this determination was based on the number of the nucleotides between the specific pair of primers for each cDNA (Fig 1). With the characterization of specific primers for m₁ to m₅ mAChRs, we then used these primers to probe the expression of mAChR subtypes in purified rat ventricular myocytes. RT-PCR was performed on total RNA obtained from 2 to 3 million purified ventricular myocytes and on freshly isolated individual myocytes. Fig 2 shows the amplification of the cDNA using RT-PCR from total RNA (lanes 2 to 8) and single-cell PCR (lanes 9 to 12). The mAChR DNA bands corresponding to m₁ (lane 2) and m₂ (lane 4) were amplified, whereas those corresponding to m₃, m₄, and m₅ (lanes 6 to 8) were not. Both m₁ and m₂ PCR products migrated in the gel according to their expected molecular weights (see "Materials and Methods"). Note that no PCR products were detected when RT was omitted from the RT-PCR reaction, demonstrating that the amplified products are indeed from cDNA and not from genomic DNA (lanes 3 and 5).

View larger version (47K): [in this window] [in a new window]   Figure 1. Gel electrophoresis of RT-PCR products amplified from mouse brain: The effectiveness of the primers was checked by amplifying all five mAChR sequences from mouse brain. A DNA standard marker is shown in lane 1 of the gel. Messages for m1 to m5 mAChR subtypes were evident (lanes 2 to 6, respectively).

View larger version (34K): [in this window] [in a new window]   Figure 2. Gel electrophoresis of RT-PCR products amplified from total RNA and from single-cell PCR. The RT-PCR was carried out as described in "Materials and Methods." DNA products from PCR were resolved on 1.2% agarose gel and visualized by ethidium bromide staining. Size markers are indicated on both sides of the gel, with bands labeled in base pairs. Total RNA RT-PCR is shown in lanes 2 to 8: Messages for the m1 (lane 2) and m2 (lane 4) mAChR subtypes from total RNA were apparent. The negative RT-PCR control for m1 and m2 mAChRs, run in parallel, contained all the components except RT (lanes 3 and 5, respectively). No DNA bands for m3 (lane 6), m4 (lane 7), and m5 (lane 8) were amplified. Single-cell PCR is shown in lanes 9 to 12: Messages for m1 (lane 12) and m2 (lane 10) were amplified. Lanes 11 and 9 are negative controls for m1 and m2 mAChRs, respectively.

Although total RNA was obtained from Percoll-purified freshly dissociated ventricular myocytes, this preparation could be contaminated with other kind of cells, eg, fibroblasts, smooth muscle cells, or nerve endings. When the purified cell preparation was viewed through a Nikon inverted microscope (magnification, x400), we did not see any contaminating cells in the preparation. However, when these cells were cultured in Joklik's medium for 16 to 20 hours, fibroblasts (1% to 2%) were seen in the culture. Even though the number of the contaminating fibroblasts was <2%, the presence of these cells could contribute to false-positive results, as some of these cells are known to possess mAChRs other than M₂.³¹ To avoid this problem, we repeated RT-PCR on individual freshly dissociated ventricular myocytes (single-cell PCR). Ventricular myocytes, profiled individually for the five subtypes of mAChRs, uniformly displayed mRNA for m₁ and m₂ mAChRs (lanes 12 and 10 of Fig 2, respectively; n=5). None of the myocytes profiled expressed messages for m₃ to m₅ mAChRs (data not shown).

Validation of the PCR Products
To confirm the identity of the PCR products, the amplified DNAs from total RNA were subjected to restriction digestion and Southern blot hybridization. Ava I, Sau3AI, and Pvu II were selected for m₁ mAChR restriction digestion. On the basis of known restriction sites (DNA Strider, version 1.1) within PCR products, each of these enzymes was expected to produce two new fragments of known molecular weights. The fragment sizes expected from these restriction enzymes were as follows: Ava I, 95 and 246 bp; Sau3AI, 97 and 244 bp; and Pvu II, 157 and 184 bp. As shown in Fig 3, digestion of the 341-bp fragment resulted in the fragments expected for m₁ mAChR. Similarly, the amplified PCR product for m₂ mAChR revealed unique restriction sites for NspBII, Apa I, and Ban II enzymes specific for m₂ mAChR (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 3. Restriction fragments of PCR products. The 341-bp (m1) PCR product was digested with restriction enzymes specific for the m1 mAChR sequence. The resulting fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. Ava I, Sau3AI, and Pvu II were used individually to digest the m1 mAChR–amplified fragment (lanes 2, 3, and 4, respectively). Lane 1 shows the uncut m1 341-bp band. A DNA standard marker is shown at the left of the gel, and the numbers on its left indicate the size of DNA markers in base pairs.

Products of RT-PCR were further validated by Southern blot hybridization with a ³²P-labeled 1.8-kb DNA fragment coding for mouse m₁ mAChR. (Fig 4). The DNA probe for m₁ mAChR consistently hybridized only to its PCR product (341 bp). This further confirmed the presence of m₁ mAChR in the rat ventricular myocytes. The m₁ probe did not hybridize to m₂ mAChR PCR products (Fig 4).

View larger version (23K): [in this window] [in a new window]   Figure 4. Southern blot of RT-PCR products. Each sample containing 10 µL PCR product was separated on a 1.5% agarose gel. After electrophoresis, the DNA was transferred to a nitrocellulose membrane. A 1.8-kb DNA fragment encoding mouse m1 muscarinic receptor protein was used as the template. The conditions for hybridization were as stated in "Materials and Methods." The DNA probe for m1 mAChR consistently hybridized only to its PCR product. The probe did not hybridize with the m2 mAChR PCR product.

To confirm that the identified mAChR mRNA are translated into proteins that are expressed on the surface membranes of the cardiac myocytes, subtype-specific antibodies (m₁ to m₃) were used in immunofluorescence studies to probe for the presence of mAChR proteins. The results obtained indicated a diffuse fluorescent staining pattern for cells incubated with m₁ and m₂ mAChR antibodies (Fig 5B and 5C). The diffuse nature of the staining is probably due to the fact that these are low-abundance proteins and also to the nonconfocal nature of the image. No fluorescence was detected in cells in which there was no antibody present or in cells incubated with m₃ mAChR antibody (Fig 5A and 5D). In the latter case, however, there appeared to be some artifactual labeling of the nucleus.

View larger version (71K): [in this window] [in a new window]   Figure 5. Immunofluorescent detection of mAChR subtypes. The identities of the m1 to m3 mAChR subtypes on the surface membranes of the ventricular myocytes were probed with subtype-specific antibodies. In control cells (A, no primary antibody) and in cells incubated with m3 mAChR antibody (D), no significant fluorescence was detected. However, myocytes incubated with m1-specific (B) and m2-specific (C) antibodies showed significant fluorescence, indicating the presence of m1 and m2 mAChR subtype proteins.

Effects of Muscarinic Stimulation on Ca²⁺ Transients
Carbachol induces a positive inotropic effect by stimulation of low-affinity mAChRs.^{4 32} It has also been demonstrated that m₁ mAChRs are functionally coupled to the stimulation of phosphoinositide hydrolysis,^{19 33} and activation of these receptors in guinea pig heart produces a positive inotropic effect by increasing the amplitude of the L-type Ca²⁺ current.¹⁹ We have previously demonstrated that 300 µmol/L carbachol increased basal [Ca²⁺]_i in unstimulated rat ventricular myocytes.³⁴ Therefore, we investigated whether the effects of carbachol on Ca²⁺ fluxes in isolated cardiac cells are mediated through m₁ mAChRs.

The effects of a high concentration of carbachol (300 µmol/L) were investigated in PTX-treated and untreated rat ventricular myocytes. PTX treatment was performed to functionally inactivate the inhibitory effects of m₂ mAChR subtypes on Ca²⁺ transients. In 8 of 10 cells pretreated with PTX, carbachol enhanced the amplitude of Ca²⁺ transients (Fig 6). Fig 6, left, shows the time course of the effect of carbachol and pirenzepine on Ca²⁺ transients. Carbachol increased the basal Ca²⁺ level from 96±7 to 118±8 nmol/L and the peak Ca²⁺ level from 519±32 to 640±36 nmol/L (n=8) (Fig 6, right). Carbachol also produced a similar increase in the amplitude of Ca²⁺ transients in myocytes that were not treated with PTX; however, under these conditions, this response was only seen in 2 of 10 cells.

View larger version (67K): [in this window] [in a new window]   Figure 6. Effect of carbachol (CCh) on Ca2+ transients in PTX-treated rat heart cells. Isolated heart cells were pretreated with 100 ng/mL PTX at room temperature for 16 hours. Ca2+ transients were first produced by field stimulation in control conditions at 1 Hz. CCh (300 µmol/L) increased both diastolic and the peak Ca2+ levels. Left, Time course of the response of the Ca2+ transients to CCh and pirenzepine (PZP). Right, Fura 2 340/380-nm fluorescence ratio calibrated in terms of cytosolic free Ca2+. Single traces of the intracellular Ca2+ transients were obtained under control conditions () and in the presence of CCh () and PZP ().This effect of carbachol was reversed by 10 nmol/L PZP.

We investigated the ability of pirenzepine and methoctramine to antagonize the increase in the magnitude of Ca²⁺ transients induced by carbachol. These compounds are selective blockers for m₁ and m₂ mAChRs, respectively,³⁵ given that they are the only subtypes present in these cells. The increase in peak Ca²⁺ level produced by 300 µmol/L carbachol was reversed by 10 nmol/L pirenzepine (Fig 6, n=4). At this concentration, pirenzepine acts as a selective antagonist of the m₁ mAChR subtype.^{36 37} On the other hand, up to 100 nmol/L methoctramine failed to modify the response of PTX-treated heart cells to 300 µmol/L carbachol (n=3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of the present study are that in adult rat ventricular myocytes (1) mRNA for the m₁ and m₂ but not the m₃ to m₅ mAChR subtypes can be detected by RT-PCR at the single-cell level, (2) the encoded m₁ and m₂ mAChR proteins can be detected by indirect immunofluorescence, and (3) carbachol produces a PTX-insensitive increase in the peak intracellular Ca²⁺ transients that is antagonized by pirenzepine, a selective m₁ mAChR antagonist, but not by methoctramine, an m₂ mAChR antagonist.

Dual effects of muscarinic receptor activation on various cardiac cell preparations are well documented.^{3 38} The prevailing view among investigators in this field has for some time been that all effects of muscarinic receptor activation on the cardiac excitation and contraction can be attributed to the m₂ mAChR subtype. Evidence to support this notion was provided through receptor binding studies^{10 11} as well as through molecular studies in which Northern blot analysis of mRNA derived from rat atria revealed the presence of mRNA for just the m₂ mAChR.^{17 18} In this one-receptor scheme, the inhibitory effects of cholinergic agonists were attributed to the m₂ mAChR coupled efficiently to a PTX-sensitive G_i/G_o protein. The stimulatory effects, which require a much higher concentration of agonist (>10 µmol/L), were also thought to be mediated through the m₂ mAChR, which was either coupled less efficiently to a PTX-insensitive G protein or was not G-protein dependent.³⁹

An alternative scheme to explain the dual effects of muscarinic receptor activation on cardiac excitation and contraction is that there is another mAChR subtype expressed at low levels in heart cells that mediates the stimulatory effects of cholinergic agonists. The low level of expression of this other subtype would make its detection difficult by Northern blot and receptor binding studies because of limits to the sensitivities of these techniques.⁴⁰ In the present study, we have used the much more sensitive technique of reverse transcription followed by PCR to probe for the expression of different mAChR subtypes in freshly isolated adult rat ventricular myocytes. The myocytes used in these studies were purified to ensure that they were essentially free of contamination by other cell types, since whole heart possesses cells other than cardiomyocytes, some of which are known to possess mAChRs other than m₂.³¹ However, we realize that purifying this preparation with one-step Percoll centrifugation may not be adequate to eliminate all the contaminating cells. Thus, we also performed RT-PCR on individual myocytes. Results from single-cell PCR confirmed that these cells express mRNA only for the m₁ and m₂ mAChRs. Furthermore, the translation of m₁ and m₂ mAChR mRNA into sarcolemmal receptor proteins was confirmed by immunofluorescence studies with subtype-specific antibodies.

We next investigated whether the identified m₁ receptor was responsible for the stimulatory effects of cholinergic agonists by recording the effects of carbachol (300 µmol/L) on the size of Ca²⁺ transients in ventricular myocytes pretreated with PTX. The results indicated that carbachol produced an increase in the magnitude of Ca²⁺ transients elicited by electric field stimulation in 80% of the cells. The reason that this increase was not seen in every cell is not obvious. One plausible explanation is that the signal-transducing mechanism for m₁ mAChRs varies greatly among cells in different regions of ventricle (left versus right ventricle, endocardium versus epicardium). It is well known that the parasympathetic innervation of the heart shows a significant regional difference.^{1 2} As a result, cells from different areas of the heart may respond to carbachol differently because of a difference either at the receptor level or at the signal transduction level.

Our results further indicated that the carbachol-induced increase in cytosolic Ca²⁺ transients was antagonized by the m₁-selective antagonist pirenzepine at a concentration (10 nmol/L) that does not block the m₂-mediated inhibitory effect of carbachol. In contrast, the m₂-selective antagonist methoctramine was without effect on the stimulatory response up to a concentration of 100 nmol/L. These results suggest that the stimulatory responses of cholinergic agonists on rat ventricular myocytes are mediated through an m₁ receptor coupled to a PTX-insensitive G protein. This result is in agreement with the recent demonstration by Gallo et al¹⁹ that high concentrations of carbachol could directly stimulate the L-type Ca²⁺ current in PTX-treated guinea pig ventricular myocytes via activation of m₁ mAChRs. Such an effect could be responsible for the carbachol-induced increase in the amplitude of Ca²⁺ transients in PTX-treated rat ventricular myocytes reported in the present study. The present data, however, appear to be inconsistent with other reports. Specifically, Matsumoto and Pappano⁴¹ have described an inward Na⁺ current activated by high concentrations of carbachol in guinea pig ventricular myocytes, which they postulated to underlie the muscarinic agonist–induced positive inotropic response in this preparation. On the basis of pharmacological studies with the m₁ and m₂ mAChR antagonists, pirenzepine and AF-DX 116, carbachol was assessed to induce this inward Na⁺ current through activation of m₂ mAChRs in a PTX-insensitive manner.⁴² It is conceivable that both the PTX-insensitive m₁ and m₂ mAChR pathways may coexist in cardiac myocytes and mediate the stimulatory effects of muscarinic agonists on the inotropic and chronotropic state of the heart. In this regard, the m₁ mAChR pathway would be predicted to be important for mediating a positive inotropic response by enhancing Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum through its increase of the L-type Ca²⁺ current. On the other hand, the carbachol-induced m₂ mAChR–mediated inward Na⁺ current may be important for generating a positive chronotropic response by depolarizing the cell and thus shifting the membrane potential closer to the threshold potential. Also, this pathway may contribute to the positive inotropic effect induced by muscarinic agonists by inducing an increase in [Ca²⁺]_i via activation of the Na⁺-Ca²⁺ exchanger.⁴³ Such a mechanism would be consistent with previous work in which we have shown that carbachol increases the basal [Ca²⁺]_i in quiescent rat heart myocytes.³⁴ This carbachol-induced increase in [Ca²⁺]_i was dependent on Ca²⁺ influx from the extracellular medium and was abolished in Na⁺-free solution. Although no attempt was made to identify which subtype of mAChR is responsible for this increase in [Ca²⁺]_i, these data were interpreted as suggesting that a Na⁺-dependent process was the primary event underlying muscarinic receptor–mediated influx of Ca²⁺ in quiescent rat heart myocytes. Finally, it has been shown that activation of transfected m₂ mAChRs can cause turnover of phosphoinositides through activation of phospholipase C by G-protein ß subunits.⁴⁴ This activation could partially account for the m₂ mAChR–induced stimulatory effects on the heart. It should be noted, however, that this process is PTX sensitive.⁴⁴

The present study is part of a growing body of evidence that challenges the view that the dual effects of cholinergic agonists are mediated through just the m₂ mAChR. The present results appear to be best explained by a model in which the stimulatory effects of muscarinic agonists on the heart are mediated through m₁ mAChRs expressed on the surface membranes of cardiac cells at a much lower level than m₂ mAChRs. The existence of a smaller number of m₁ mAChRs in heart could be of physiological importance. The stimulant effects by m₁ mAChRs may serve as a "reflex" or "compensatory" mechanism to limit the inhibitory effects on the heartbeat by m₂ mAChRs during vagus nerve stimulation.²

	Selected Abbreviations and Acronyms

 

GPBS = PBS containing 5% goat serum mAChR = muscarinic acetylcholine receptor PCR = polymerase chain reaction PTX = pertussis toxin RT = reverse transcriptase

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-33333, DA-00464, and NS-30454 and by Grant-in-Aid 94-421 from the American Heart Association, New York State Affiliate, Inc. Dr Colecraft is a predoctoral fellow of the Pharmaceutical Research and Manufacturers of America Foundation. Dr Wang is a postdoctoral fellow of Cardiovascular Research Training Program 2-T32-HL-07220. We wish to thank our colleagues Drs A.W. Tank and Lisa Rubin for helpful discussions.

Received January 9, 1996; accepted March 27, 1996.

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