©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
MMuscarinic Receptors Heterologously Expressed in Cardiac Myocytes Mediate Ras-dependent Changes in Gene Expression (*)

M. Teresa Ramirez (§) , Ginell R. Post (¶) , Prakash V. Sulakhe (**) , Joan Heller Brown (§§)

From the (1) Department of Pharmacology and Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California 92093

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of -adrenergic receptors in neonatal ventricular cardiomyocytes induces hypertrophic changes including activation of the atrial natriuretic factor (ANF) gene. This receptor couples to Gto activate phospholipase C (PLC) and protein kinase C, which have been implicated as mediators of the hypertrophic response. To directly determine whether receptor coupling to G/PLC is sufficient to induce ANF expression, we expressed wild-type and chimeric muscarinic cholinergic receptors (mAChRs) with altered G-protein coupling properties in cardiac myocytes and examined their ability to activate an ANF promoter/luciferase reporter gene. The cholinergic agonist carbachol failed to induce transcriptional activation of the ANF reporter gene through endogenous G-linked MmAChRs or in cells transfected with MmAChRs. In contrast, in cells transfected with MmAChRs, which effectively couple to G/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as determined by immunofluorescence. Carbachol-mediated ANF gene expression was inhibited by the mAChR antagonist pirenzepine with a Kvalue characteristic of an MmAChR. Studies using chimeric M- and MmAChRs demonstrated that the N-terminal 21 amino acids of the third intracellular loop of the MmAChR were required for receptor coupling to ANF gene expression. This region, previously shown to specify receptor coupling to G/PLC, also conferred partial activity to a chimeric Mreceptor. We further demonstrated that MmAChR coupling to ANF gene expression was Ras-dependent since co-expression of dominant-interfering Ala-15 Ras inhibited MmAChR-induced ANF expression by 60%. In contrast, ANF expression induced by the chimeric Mreceptor was not blocked by dominant-interfering Ras. We suggest that receptor coupling to G/PLC is sufficient to induce ANF expression and that a Ras-dependent pathway contributes additional signals required for maximal MmAChR-mediated ANF gene expression.


INTRODUCTION

Cultured neonatal ventricular cardiomyocytes are a useful model for studying changes in gene expression associated with myocardial cell hypertrophy. The hypertrophic response, characterized by an increase in cell size, is accompanied by up-regulation of the myosin light chain-2 (MLC-2)() gene and reactivation of the atrial natriuretic factor (ANF) and other genes (1, 2, 3, 4, 5) . Signals that lead to cardiac cell growth and gene expression can be generated through activation of G-protein-coupled -adrenergic receptors (AdrRs) (6, 7) . In cardiac cells, the AdrR stimulates phospholipase C (PLC) through the guanine nucleotide (GTP)-binding protein G(8, 9) and activates protein kinase C (10, 11) . Both Gand protein kinase C have been implicated as mediators of the genetic response induced by AdrR activation (9, 12, 13, 14, 15) . In addition Ras, a small GTP-binding protein involved in growth factor signaling pathways, has been shown to be required for AdrR-induced increases in cardiac cell size and gene expression (16) . It is not known whether an agonist needs to activate Gas well as Ras to regulate myocardial cell growth and gene expression.

In order to determine how G-protein-coupled receptors regulate cardiac gene expression, we have heterologously expressed wild-type and chimeric receptors that have altered G-protein/effector coupling properties. The Mmuscarinic cholinergic receptor (MmAChR) is a G-protein-coupled receptor that, like the AdrR, activates PLC through its interaction with G(17) . MmAChRs are not present in cardiomyocytes (18, 19) , which predominantly express MmAChRs that regulate adenylyl cyclase through interaction with G(20, 21) . The MmAChR and MmAChR are highly conserved in their transmembrane spanning regions, but vary significantly in their third intracellular (i3) loop, a region shown to confer G-protein/effector coupling specificity (22, 23, 24, 25) . The determinants for G-protein recognition and activation appear to be shared among other receptors that utilize similar signaling pathways. For example, peptides representing the i3 loop of the MmAChR have recently been shown to compete with the i3 loop of the AdrR for activation of PLC (26) , indicating that these receptors interact with G-proteins in functionally similar ways.

The studies reported here demonstrate that heterologously expressed mAChRs of the Msubtype, like endogenous AdrRs, transduce signals leading to cardiac-specific gene expression. In contrast, expression of MmAChRs does not transduce muscarinic receptor agonist binding into ANF gene expression. Using chimeric receptors, we demonstrate that a 21-amino acid region of the i3 loop critical for coupling MmAChR to G/PLC is necessary for receptor-mediated ANF expression and can induce a partial response when exchanged for the homologous region in the MmAChR. Expression of dominant-interfering Ala-15 Ras inhibits MmAChR-induced ANF gene expression but does not block the response mediated by the chimeric Mreceptor, suggesting that the 21-amino acid region conferring G-protein selectivity is not sufficient to couple the mAChR to a Ras-dependent pathway.


MATERIALS AND METHODS

Cell Culture Procedure

Neonatal rat ventricular cardiomyocytes from 1-3-day-old Sprague-Dawley rats were isolated and cultured as described previously (15, 27) . Cells were plated at a density of 3.5-4.0 10/cmon 60-mm gelatin-coated tissue culture dishes or on 25-mm etched coverslips (Bellco) and maintained overnight in 4:1 Dulbecco's modified Eagle's medium/medium 199 (Life Technologies, Inc.) containing 10% horse serum, 5% fetal calf serum, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin).

Plasmid Constructs

The following promoters fused to firefly luciferase cDNA were used as reporter gene constructs: a 638-base pair fragment of the rat ANF promoter (2) , a 2700-base pair fragment of the MLC-2 promoter (28) , and the Rous sarcoma virus (RSV) long term repeat (15) . A series of muscarinic receptors cloned into the cytomegalovirus promoter-driven (pCMV) expression vector (29, 30) were provided by Drs. Elliott Ross and Stephen Wong. These are: MmAChR (cDNA encoding the human MmAChR); MmAChR (cDNA encoding the human MmAChR); M:NM(cDNA of human MmAChR containing amino acids 208-230 from the human MmAChR); and M:NM(cDNA of human MmAChR containing amino acids 210-230 from the human MmAChR). The dominant-interfering Ala-15 Ras expressed in the pZip backbone was provided by Dr. Kirk Knowlton (9) .

Transient Transfection Assay

Myocytes were transfected in serum-containing medium using a modified calcium phosphate transfection technique as described previously (15) . A total of 8-10 µg of DNA was used, which consisted of the appropriate luciferase reporter gene (3 µg) and either the pCMV backbone or pCMV vector containing various receptor cDNAs (0.1-6 µg). Following transfection, cells were washed extensively, cultured in the presence of carbachol or phenylephrine for 48 h, and harvested in a 0.5% Triton X-100 buffer. Luciferase activity and protein concentration were determined for each sample as described (15) .

Measurement of Phosphoinositide Hydrolysis

Myocytes were labeled overnight with 3 µCi/ml [H]inositol in serum-free medium and [H]inositol phosphate formation assayed in the presence of 10 m M LiCl as described (31) .

Immunofluorescence Analysis

Indirect immunofluorescence analysis was performed using a modification of a previously described procedure (7) . Briefly, cells plated on etched coverslips were transfected with the pCMV backbone or MmAChR expression plasmid along with an RSV/luciferase reporter gene. After a 48-h treatment with carbachol or phenylephrine, cells were fixed with 3% paraformaldehyde and permeabilized with 0.3% Triton X-100. Transfected cells were identified by immunostaining with a polyclonal anti-luciferase antibody (Cortex Biochem) followed by a fluorescein-conjugated, goat anti-rabbit antibody. ANF expression was detected by using a mouse monoclonal antibody against ANF (provided by Dr. C. Glembotski, San Diego State University) and a rhodamine-conjugated, goat anti-mouse antibody. Luciferase and ANF protein expression were analyzed by fluorescence microscopy using a Plan-neofluar 40 objective (Zeiss).

Radioligand Binding Experiments

Neonatal rat cardiomyocytes were transfected with the pCMV backbone or MmAChR cDNA as described above, and membrane fractions were prepared. To determine mAChR density, membranes were incubated in a Hepes-buffered salt solution (32) with 1 n M (-)-[ N- methyl-H]QNB (a concentration 10 times its K) at 30 °C for 60 min. Nonspecific binding, determined in the presence of 1 µ M atropine, was <10% of total binding. For competition experiments, the assay contained 0.6 n M (-)-[ N- methyl-H]QNB and varying amounts of atropine (0.01 n M to 100 µ M) or pirenzepine (0.1 n M to 30 µ M). The results of competition experiments were com-puter-analyzed by using EBDA/LIGAND to obtain Kvalues for the antagonists (32) .


RESULTS

We first determined whether endogenous MmAChRs in neonatal cardiomyocytes regulate ANF gene expression by transiently transfecting cells with an ANF promoter/luciferase reporter gene and stimulating cells with the stable acetylcholine analog carbachol for 48 h. Carbachol failed to increase luciferase expression while the -adrenergic receptor agonist phenylephrine caused 10-fold activation of the ANF promoter (Fig. 1 A). Subsequent experiments examining PLC activation demonstrated that carbachol caused only modest stimulation of [H]inositol phosphate formation when compared to the robust response to phenylephrine (Fig. 1 B). This is consistent with evidence that mAChRs of the Msubtype couple poorly to PLC (23, 33, 34, 35) . Thus, endogenous cardiac mAChRs show low efficacy for activation of PLC and do not couple to effectors regulating ANF expression.


Figure 1: Differential effects of carbachol and phenylephrine treatment on ANF gene expression and phosphoinositide formation. A, neonatal rat ventricular myocytes were transfected with an ANF promoter/luciferase reporter gene and then incubated for 48 h with either no drug, 300 µ M carbachol, or 100 µ M phenylephrine (with 2 µ M propranolol to block -adrenergic receptors). Luciferase activity was normalized to micrograms of protein for each sample and agonist-induced increases expressed as -fold stimulation relative to no drug treatment. B, inositol monophosphate accumulation was assayed in [H]inositol-labeled myocytes which were treated with agonists plus 10 m M LiCl for 20 min at the concentrations tested above. Data are the mean ± S.E. of quadruplicate samples from a representative experiment.



To determine whether ANF gene expression could be induced if G/PLC-linked mAChRs or a greater number of MmAChRs were present, we transfected myocytes with the ANF reporter gene along with either the pCMV backbone vector or with human Mor MmAChR cDNA and subsequently treated cells with carbachol for 48 h (Fig. 2). In cells transfected with the vector alone or MmAChR cDNA, carbachol failed to induce ANF gene expression. However, in cells expressing the MmAChR, carbachol treatment resulted in a 10-fold increase in luciferase activity. To examine the concentration dependence of this response, cardiomyocytes were transfected with various concentrations of MmAChR cDNA. As shown in Fig. 3, concentrations of the MmAChR cDNA as low as 0.01 µg conferred maximal responsiveness to carbachol. Furthermore, the carbachol dose-response curve was shifted to the right as the amount of receptor cDNA was decreased, suggesting a relationship between the response and the number of expressed receptors (Fig. 3).


Figure 2: Transcriptional activation of ANF gene expression in myocytes transfected with wild-type muscarinic cholinergic receptors. Neonatal rat ventricular myocytes were co-transfected with the backbone pCMV expression vector or the pCMV vector containing Mor Mreceptor cDNA, along with an ANF promoter/luciferase reporter gene. Cells were then incubated for 48 h in the absence ( hatched bars) or presence ( solid bars) of 300 µ M carbachol. Luciferase activity was normalized to micrograms of protein. The data are expressed as -fold stimulation by carbachol relative to untreated (no drug) samples and represents the mean ± S.E. of triplicate or quadruplicate samples from 2-8 experiments.




Figure 3: Dose dependence of ANF gene expression on receptor and carbachol concentrations. Cardiomyocytes were co-transfected with either 0.01, 0.1, or 3.0 µg of MmAChR cDNA along with the ANF promoter/luciferase reporter gene. Cells were then incubated for 48 h with varying concentrations of carbachol. Luciferase activity was measured and normalized to micrograms of protein. The data are expressed as percent of maximal ANF luciferase measured at the highest carbachol concentration and represent the mean ± S.E. of duplicate or triplicate samples from 1-3 experiments.



Because a relatively small fraction of the cardiomyocytes express exogenous DNA (<5% based on -galactosidase staining), we could not detect an increase in mAChR expression using radioligand binding techniques. Thus, in binding experiments using a saturating concentration of [H]QNB, pCMV-transfected cells had specific [H]QNB binding of 189 ± 9 fmol/mg of protein, and MmAChR-transfected cells had essentially the same amount of specific binding (201 ± 8 fmol/mg of protein; data not shown). We were, however, able to demonstrate that heterologously expressed MmAChRs regulate ANF expression by using pharmacological characterization. Cells transfected with the MmAChR and ANF reporter gene were incubated with various concentrations of the mAChR antagonists atropine and pirenzepine prior to the addition of 10 µ M carbachol. The ICvalues for inhibition of carbachol-stimulated ANF expression were determined from the curves shown in Fig. 4and corrected to give estimated Kvalues for the antagonists. Kvalues were calculated from radioligand binding competition experiments (Fig. 4). The binding constants determined for atropine in experiments examining ANF expression and radioligand binding were similar ( K= 0.8 n M; K= 1.2 n M) while those for pirenzepine were markedly different ( K= 11 n M; K= 600 n M). The mAChR mediating ANF gene expression therefore has about a 50-fold higher affinity for pirenzepine than does the endogenous receptor assessed by radioligand binding. The estimated Kvalue for pirenzepine antagonism of ANF expression (11 n M) is characteristic of an MmAChR (36, 37, 38) .


Figure 4: Inhibition of carbachol-stimulated ANF gene expression and of [H]QNB binding by atropine and pirenzepine. Ventricular myocytes transfected with MmAChR cDNA and the ANF promoter/luciferase reporter gene were treated with increasing concentrations of either atropine ( open squares) or pirenzepine ( open circles) 30 min prior to the addition of 10 µ M carbachol. After a 48-h drug treatment, luciferase activity was assayed. Data are expressed relative to maximal carbachol-stimulated ANF luciferase measured in the absence of antagonist. Each point represents the mean ± S.E. of triplicate samples from two experiments. The ICvalues for atropine and pirenzepine (13 n M and 200 n M, respectively) were corrected by the Cheng-Prusoff equation (using an ECfor carbachol of 0.63 µ M) to give the estimated K. For radioligand binding experiments, myocyte membranes were incubated with 0.6 n M [H]QNB and varying amounts of atropine ( solid squares) or pirenzepine ( solid circles). The results shown are for the specific binding and are the mean ± S.E. of triplicate samples from two experiments. The results were analyzed by EBDA/LIGAND to give the calculated K values.



In ventricular cardiomyocytes, AdrR activation increases ANF protein (9, 16) . To determine whether carbachol induces ANF protein expression in MmAChR-transfected myocytes, cells were transfected with MmAChR cDNA or the pCMV backbone along with an RSV/luciferase expression vector and analyzed by immunofluorescence. Luciferase expression was used to identify transfected cells and ANF protein expression was examined for each experimental condition. About 60% of the cells transfected with the pCMV backbone vector showed increased ANF immunoreactivity when treated with phenylephrine, whether or not the cells expressed luciferase (). In contrast, stimulation with carbachol increased ANF expression only in myocytes transfected with MmAChR cDNA and exclusively in cells expressing luciferase. These data confirm results obtained from transient transfection experiments by demonstrating that ANF protein expression is induced by carbachol only in cells transfected with MmAChR cDNA.

The specificity of MmAChR-mediated gene expression was further investigated by examining the transactivation of the cardiac-specific MLC-2 promoter and the nonspecific viral (RSV) promoter/luciferase reporter genes. In cells transfected with MmAChR cDNA, carbachol increased MLC-2 gene expression 12-fold (Fig. 5). In contrast, carbachol did not activate the RSV promoter in these cells. These results indicate that activation of expressed MmAChRs does not have a nonspecific effect on gene expression but is selectively coupled to activation of at least two cardiac genes known to be induced during myocardial cell hypertrophy.


Figure 5: Effect of M muscarinic cholinergic receptor activation on cardiac-specific and nonspecific gene expression. Neonatal rat ventricular myocytes were transfected with expression constructs containing luciferase reporter genes driven by either the ANF, MLC-2, or RSV promoter along with the wild-type MmAChR. Cells were incubated without ( hatched bars) or with ( solid bars) 300 µ M carbachol for 48 h, and luciferase activity was determined and normalized to micrograms of protein. The data are expressed as -fold stimulation by carbachol relative to untreated (no drug) samples and represents the mean ± S.E. of triplicate or quadruplicate samples from 1-3 experiments.



To examine regions of the receptor responsible for transducing signals leading to cardiac gene expression, we tested chimeric receptors in which regions of the i3 loop were altered. Initially, we tested a chimeric M:IIreceptor in which the entire i3 loop and part of the i2 loop of the Mreceptor were replaced with those of the -adrenergic receptor (29, 30) . This chimeric receptor failed to transduce carbachol binding into increased ANF gene expression at any cDNA concentration tested (39) . Similarly, a chimeric M:NMreceptor which differs from the native MmAChR by replacement of the N-terminal 21 amino acids of the i3 loop with the homologous region of the MmAChR was unable to induce ANF expression even at a concentration 60 times that of the wild-type MmAChR (compare 0.1 µg of M versus 6 µg of M:NMin Fig. 6 ). Both of these chimeric Mreceptors are expressed at levels similar to those of the wild-type receptor in COS or A293 cells (29) .() We also tested a chimeric M:NMreceptor in which the N-terminal 23 amino acids of the MmAChR i3 loop are replaced with those of the MmAChR i3 loop (Fig. 6). Activation of this chimeric receptor with carbachol significantly increased ANF gene expression, although the maximal response was 50% of that seen with the MmAChR even at the highest concentrations of receptor cDNA (6 µg) or carbachol (300 µ M) tested. These data demonstrate that as little as 21 amino acids from the MmAChR confers receptor specificity for coupling to ANF gene expression.


Figure 6: Effects of wild-type and chimeric muscarinic cholinergic receptors on ANF gene expression. Ventricular myocytes were transfected with cDNA for MmAChR (0.1 or 3 µg), MmAChR (3 or 6 µg), M:NMchimera (3 or 6 µg), or M:NMchimera (0.1, 3, or 6 µg) together with the ANF luciferase reporter gene. Cells were stimulated with 10 µ M carbachol for 48 h, and luciferase activity was measured and normalized to micrograms of protein. The data are expressed as -fold stimulation relative to no drug treatment and represent the mean ± S.E. of triplicate samples from 2-3 experiments. The same results were obtained when cells were stimulated with 300 µ M carbachol.



Ras has been shown to be involved in AdrR-mediated ANF gene expression (9, 16) . To determine whether Ras was similarly involved in mAChR-mediated ANF gene expression, we transiently co-expressed either the Mor chimeric M:NMreceptor cDNA along with a dominant-interfering Ala-15 Ras cDNA to block Ras function. MmAChR-mediated ANF gene expression was significantly inhibited (60%) by the dominant-interfering Ras (Fig. 7). Thus, Ras appears to be a mediator of MmAChR-induced ANF expression, as previously shown for the AdrR. In contrast, ANF expression induced by the chimeric M:NMreceptor was not inhibited by the dominant-interfering Ras. Therefore, the 21 amino acids of the MmAChR i3 loop appears to specify coupling to G/PLC but not to a Ras-dependent pathway.


DISCUSSION

Heterologous expression of wild-type and chimeric G-protein-coupled receptors provides a new approach for studying signaling pathways that mediate hypertrophic changes in gene expression in neonatal ventricular myocytes. This approach can be used because the functional response of interest, induction of the cardiac-specific ANF gene, can be monitored in transiently transfected cells using a sensitive ANF promoter/luciferase reporter gene and by immunofluorescent detection of ANF protein. The MmAChR is a structural and functional homolog of the endogenous AdrR which, we reasoned, would mimic AdrR-mediated hypertrophic responses and which could be experimentally modified to evaluate the functional determinants required for coupling receptors to gene expression.

In order to demonstrate that transcriptional activation of the ANF gene by carbachol was occurring through the heterologously expressed MmAChRs, we first showed that carbachol-stimulated ANF gene expression was blocked by the muscarinic receptor antagonist pirenzepine with a Ksimilar to that determined for MmAChRs in other systems (36, 37, 38) . We also demonstrated that the dose-response curve for carbachol shifts to higher concentrations as the amount of transfected MmAChR cDNA is decreased (Fig. 3), suggesting that the response depends on the number of heterologous receptors expressed. Finally, only individual myocytes that expressed exogenous cDNA showed increased ANF immunoreactivity in response to carbachol (). These data indicate that exogenous MmAChRs are expressed and generate signals that lead to transcriptional activation of the ANF gene.

Muscarinic cholinergic receptors have been extensively characterized in terms of their specificity for coupling to PLC and adenylate cyclase. Receptors of the Mand Msubtypes preferentially couple to the regulation of PLC via the pertussis toxin-insensitive G protein G, whereas M- and MmAChRs inhibit adenylate cyclase through interaction with G(21, 40, 41) . Specificity for effectors is largely conferred by the third intracellular loop of the G-protein-coupled receptor (22, 23, 24, 25) . We demonstrate that there is a clear pattern of selectivity in the requirements for coupling mAChR to ANF gene expression since the MmAChR, which couples to G/PLC, is effective while the endogenous and heterologously overexpressed G-linked MmAChRs are not (Figs. 2 and 6).

The loss of function seen with the chimeric M:IIand M:NMreceptors in cardiomyocytes is further evidence that receptor coupling to G/PLC is required for ANF expression, because these and homologous M:NMchimeras are ineffective at stimulating PLC through G-dependent pathways (23, 24, 29) .Thus, there appears to be a high degree of selectivity and little promiscuity in mAChR receptor subtype coupling to the regulation of ANF gene expression.

Remarkably, a 21-amino acid fragment was sufficient to allow the Mreceptor to function like an MmAChR in regulating ANF gene expression. This finding is consistent with the observation that this region is critical for conferring specificity for receptor coupling to PLC (21, 40) . The M:NMchimera, however, showed only about one-half the efficacy of the wild-type receptor for increasing ANF expression, even at the highest concentration tested. This could reflect a lower efficacy of the N-terminal i3 loop chimera for PLC activation, as shown for M:NMreceptors stably expressed in A9L cells (24) ; however, the M:NMand homologous M:NMchimeras are as effective as the wild-type mAChRs for inducing inositol phosphate formation in transiently transfected COS and A293 cells (23, 30) .An alternative possibility is that the M:NMchimeric receptor has a submaximal effect on ANF expression because, while it couples efficiently to G/PLC, it lacks regions needed for coupling to additional downstream effectors. The observation that the dominant-interfering Ras significantly inhibited Mbut failed to block M:NMreceptor-mediated ANF gene expression suggests that the chimeric receptor lacks the ability to couple to Ras-signaling pathways.

The role of Ras in AdrR-mediated myocardial hypertrophy has been previously demonstrated by the ability of a dominant-interfering Ras expression vector to block phenylephrine-mediated increases in cell size and ANF gene expression (16) . It has also been shown that Ras is activated in response to AdrR stimulation in cardiac myocytes (42, 43) . We report here that mAChRs must interact with a G-dependent pathway to induce transcriptional activation of ANF and that Ras function is also required for maximal ANF gene expression in response to MmAChR stimulation. While it is not possible to directly demonstrate that the heterologously expressed MmAChRs activate Ras in cardiomyocytes, the same receptor stably expressed in fibroblasts has been shown to activate Ras (44) . We are currently exploring the use of adenoviral vectors to increase the transfection efficiency of cardiac myocytes so that biochemical responses such as Ras and G/PLC activation can be directly determined in cells expressing wild-type and chimeric mAChRs. Further studies using expression of heterologous receptors should provide insight into the question of how pathways initiated by G-protein-coupled receptors collaborate with those involving Ras to regulate cardiac cell growth and gene expression.

  
Table: MmAChR-transfected cells express ANF protein

Neonatal ventricular myocytes were co-transfected with an RSV/luciferase expression plasmid along with either the pCMV backbone or MmAChR cDNA. Cells were subsequently stimulated with 300 µ M carbachol or 100 µ M phenylephrine (with 2 µ M propranolol) for 48 h. Transfected cells were identified by their expression of luciferase using an anti-luciferase antibody and a fluorescein-conjugated secondary antibody. ANF protein expression was detected with an antibody against ANF and a rhodamine-conjugated secondary antibody. The percent of ANF positive cells was calculated by dividing the number of ANF-expressing cells either by the number of cells that also express RSV/luciferase cDNA (luciferase-positive) or by the number of cells that do not express luciferase (luciferase-negative). Approximately 150 luciferase-negative and 30 luciferase-positive cells were scored for ANF immunoreactivity for each experimental condition.



FOOTNOTES

*
This research was supported in part by National Institutes of Health Research Grants HL28143 and HL46345 and a Tobacco Related Disease Research Program Grant (to J. H. B.). 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.

§
American Heart Association-California Affiliate predoctoral fellow.

Supported by National Institutes of Health Postdoctoral Training Grant HL07444.

**
Supported by grants received from the Medical Research Council of Canada and from the Heart and Stroke Foundation of Saskatchewan. Permanent address: College of Medicine, Dept. of Physiology, University of Saskatchewan, Saskatoon S7N 0W0, Canada.

§§
To whom correspondence and reprint requests should be addressed. Tel.: 619-534-2595; Fax: 619-534-4337.

The abbreviations used are: MLC-2, myosin light chain-2; ANF, atrial natriuretic factor; RSV, Rous sarcoma virus; PLC, phospholipase C; mAChR, muscarinic cholinergic receptor; AdrR, -adrenergic receptor; QNB, (±)-3-quinuclidinyl benzoate.

E. Ross, personal communication.


ACKNOWLEDGEMENTS

We thank Elliott M. Ross, Stephen K.-F. Wong, and Kirk U. Knowlton for supplying plasmids and advice concerning experimental protocols and David Goldstein for technical assistance.


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