The ala-Adrenergic Receptor Subtype Mediates Biochemical, Molecular, and Morphologic Features of Cultured Myocardial Cell Hypertrophy*

al-Adrenergic agonists induce a hypertrophic phe- notype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stim- ulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which cyl-adrenergic receptor subtype mediates the acquisi- tion of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log Ki = -8.7) and (+)-niguldipine (log Ki = -10.6). The a-adrenergic receptor-induced increase in transcriptional activation of an ANF luciferase reporter gene is inhibited over the same range of concentrations of 5-methylurapidil (log K; = -8.2) and (+)-niguldipine (log K; = -11.2) that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies a-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurap-idil (log Ki = -8.0) and (+)-niguldipine (log -10.6). In contrast, treatment with the alB selective antagonist chlorethylclonidine at a concentration of 10 PM had no effect on the adrenergically mediated induc- tion of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alA-adrenergic receptors mediate not only the early effects of al-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using al-ad-renergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, GTP proteins, is the concentration of ligand or agonist used and Kd is its affinity as determined in our binding studies. The ECso values determined for phenylephrine-stimulated phosphoinositide hydrolysis and activation of ANF luciferase expression were similar to the Kd determined for phenylephrine in the binding studies. IC, was calculated using iterative nonlinear regression analysis using the InPlot program (GraphPAD Software, San Diego, CA).

al-Adrenergic agonists induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stimulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which cyl-adrenergic receptor subtype mediates the acquisition of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log  that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies a-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurapidil (log Ki = -8.0) and (+)-niguldipine (log Ki = -10.6). In contrast, treatment with the alB selective antagonist chlorethylclonidine at a concentration of 10 PM had no effect on the adrenergically mediated induction of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alA-adrenergic receptors mediate not only the early effects of al-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using al-adrenergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, e.g. GTP binding proteins, HL-45069, HL-46435, and HL-36139 (to K. R. C.), HL-28143 and * This work was supported by National Institutes of Health Grants , and HL-02618 (to K. U. K.), American Heart Association Grants 88-0235 and 91-022170 (to K. R. C.) and AHA-CA-91-144 (to K. U. K.), and Deutsche Forschungsgemeinschaft Grant DFG Mi 294/2-1 (to M. C. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ll Representing the AHA-Bugher Foundation, Center for Molecular Biology.
should provide a powerful means of assessing their role in the induction of the molecular and morphologic parameters of myocardial cell hypertrophy.
Activation of cardiac a,-adrenergic receptors leads to rapid changes in contractility, electrophysiological properties, and metabolic responses of the myocardium (1). a,-Adrenergic agonists stimulate cardiac phosphoinositide metabolism (a), activate glycogenolysis (3), induce positive inotropic effects which are related either to an increase in free cytosolic Ca2+ levels (4) or to an increase in the responsiveness of myofilaments to Ca2+ ( 5 ) , and elicit a cascade of other acute effects on cardiac intermediary metabolism (6).
In addition to these relatively acute effects, recent studies have suggested that a-adrenergic stimuli also have long term effects on cardiac structure and function. Chronic exposure of cultured neonatal rat ventricular muscle cells to a-adrenergic agonists leads to the acquisition of several genetic and morphologic features of hypertrophy. These include induction of a distinct immediate early gene program, appearance of markers of an embryonic program of gene expression, accumulation and assembly of constitutively expressed contractile proteins into sarcomeric units, and an increase in cell size without concomitant proliferation (7,8). Activation of a subset of constitutively expressed contractile proteins and markers of an embryonic program of gene expression is largely due to selective transcriptional activation (9). The precise signaling pathways which mediate these chronic effects of a-adrenergic stimulation on cardiac ventricular muscle cells are unknown. Recent studies have implicated protein kinase C (10)(11)(12), the Ras protooncogene (13), and the GTP binding protein, G, (14), in the signaling pathways which activate genetic markers of the hypertrophic response. A detailed analysis of the a-adrenergic signalingpathways which directly or indirectly interact with these proximal signals to activate distinct features of myocardial cell hypertrophy would be facilitated by the identification of the precise a-adrenergic receptor(s) that mediate this chronic effect.
Recent studies have revealed that a,-adrenergic receptors comprise a family of closely related receptor subtypes (15,16), which regulate cellular responses through diverse signaling pathways (17). Both the alA-and alB-adrenergic receptor subtypes are expressed in rat heart as assessed by radioligand binding (18,19). Northern blotting with specific al-adrenergic receptor subtype cDNA probes demonstrates that two distinct al-adrenergic receptors are expressed in rat heart (20). The functional presence of other members of the a,-adrenergic receptor family has not been detected in rat heart. The multitude of cardiac responses to cyl-adrenergic stimulation such as its effect on inotropy, phosphoinositide hydrolysis, and gene expression raises the question as to whether distinct receptor subtypes mediate these individual responses. It is possible that activation of multiple ventricular muscle a1adrenergic receptor subtypes is required to effect integrated cellular responses that occur in response to a-adrenergic receptor agonists, Alternatively, a single al-adrenergic receptor subtype might mediate the acquisition of many of the well characterized features of the hypertrophic response.
In previous studies, identification of ml-adrenergic receptor subtype-dependent signaling pathways has focused on the relatively acute effects of a,-adrenergic stimulation on phosphoinositide metabolism or Ca2+ mobilization, using tissue slices, cultured cell lines, or surrogate cell lines which stably express cloned a,-receptor subtype cDNAs (21-23). Since & Iadrenergic stimulation of ventricular muscle cells leads to transcriptional activation of well defined genetic markers of the hypertrophic response, as well as structural/morphologic changes which can be distinguished at a single cell level, the myocyte model system may ultimately allow the implementation of genetic approaches to identify signaling pathways that lead to long term phenotypic alterations which are specific for distinct al-adrenergic receptor subtypes.
The current study provides direct evidence that a pharmacologically identified alA-adrenergiC receptor subtype activates biochemical, genetic, and morphologic features of adrenergically mediated ventricular cell hypertrophy, thereby suggesting that the entire repertoire of the hypertrophic phenotype can be activated through an alA-adrenergic receptor-dependent pathway. Identification of the ma-adrenergic receptor as the adrenergic receptor responsible for hypertrophy in cultured cardiac ventricular muscle cells, coupled with the availability of well defined genetic markers of this important adaptive cardiac muscle response, will allow genetic approaches to characterize the potentially complex interaction of the CxlA-adrenergiC receptor with other proximal signaling molecules such as the GTP binding proteins, G,, and Ras, in cardiac muscle cells.

MATERIALS AND METHODS
Cell Culture-Neonatal rat ventricular cells were cultured as previously described with minor modifications (24, 25). Briefly, hearts from 1-2-day-old Sprague-Dawley rats were recovered, atria were removed, and the ventricles were pooled and trisected. Myocytes were dispersed by digestion with collagenase I1 (Worthington) and pancreatin (GIBCO). Myocardial cell suspensions were centrifuged through Percoll step gradients to obtain cell preparations with >95% myocytes, as assessed by immunofluorescence with MLC-2 antisera (24). Myocytes were plated at a density of 3.0 X lo4 cells/cm*, in either 100-or 150-mm tissue culture dishes precoated with gelatin, in 4:l Dulbecco's modified Eagle's medium/medium 199 (GIBCO), supplemented with 10% horse serum, 5% fetal calf serum, and antibiotics (ampicillin at 34 pg/ml and gentamicin at 3 pg/ml). Following a 24h incubation in serum-containing medium, cells were washed with and incubated in serum-free medium in the presence or absence of the indicated al-adrenergic agonists and antagonists. 0.1% bovine serum albumin was added to prevent nonspecific adsorption of antagonists to plasticware. Transfected cells were treated as described below.
Transfection of Myocardial Cells-Cells were plated in 100-mm gelatinized tissue culture dishes at a density of 3.0 X lo4 celIs/cm2 and allowed to attach for 18-20 h after plating. The cells were transfected with 6 pg of pON249 and 20 pg of pANF638L utilizing a modified calcium phosphate precipitation method (26). After washing, the cells were maintained in 4:l Dulbecco's modified Eagle's medium/ medium 199 in the presence or absence of the indicated adrenergic agonist and antagonist until harvested 38-48 h later. Luciferase and 0-Galactosidase Assays-Transfected cells were washed twice with PBS' without Ca2+ or M e and then harvested in 1.0 ml of extraction buffer (100 mM Tricine, 10 mM MgSO4, 2 mM EDTA, pH 7.8, 1 mM dithiothreitol). Cells from each dish were collected by centrifugation, resuspended in 250 pl of extraction buffer, lysed by five cycles of freezing in dry ice, and thawing at 37 'c, and cell debris was removed by centrifugation. 25-pl samples from the extracts were combined with 275 rl of 73 p M luciferin (Analytical Luminescence Laboratory) and 2 mM ATP (Sigma) in extraction buffer. Luciferase activity (27) was measured in triplicate in a Monolight 401 luminometer (Analytical Luminescence Laboratory). 0-Galactosidase activity was assayed on 50-pl samples as described (28). Luciferase activities were normalized to their corresponding P-galactosidase activities. Immunofluorescence Techniques-Indirect immunofluorescence assays were performed by a minor modification of a previously described procedure (29). Briefly, the myocardial cells were grown on Lab-Tek plastic chamber slides precoated with 4 pg of laminin (Sigma) per cm2. 48 h after incubation in the indicated medium with or without al-agonists and antagonists, the cells were rinsed with PBS without Ca2+ or M e and fixed for 15 min at room temperature with 3% paraformaldehyde in 10 mM sodium phosphate, 150 mM NaCI, 1 mM MgCl,, pH 7.4. The cells were then incubated in 50 mM NH4Cl for 10 min, washed twice with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature, followed by three additional washings with PBS. Chamber slides were incubated with 1% bovine serum albumin for 10 min to block nonspecific sites, incubated with TrpE/MLC-2 antisera (24) for 60 min at 37 "C, rinsed, and washed four times with PBS. Subsequently, the chamber slides were incubated for 10 min in a 1:20 dilution of normal goat serum (TAGO, Inc.) in PBS and then incubated for 60 min at 37 "c with fluorescein-conjugated affinity-purified goat anti-rabbit IgG (Cappel Laboratories). The slides were subsequently rinsed and then washed four times with PBS, mounted on glass coverslips with 15% (w/v) Airvol 205 polyvinyl alcohol (Air Products and Chemicals, Inc.), 33% glycerol, and 0.1% sodium azide in PBS, pH 7.4, and viewed with fluorescence microscopy. Cellular areas were determined by planimetry of individual cell areas in high power fields.
Plasmid Constructs-To identify cis sequences which mediate aadrenergic-inducible expression of the ANF gene, a rat ANF promoter luciferase expression vector was created. The ANF638L chimeric reporter was constructed as described (9) by ligating an EcoRI (-638)/ Sau3a (+62) fragment of the rat ANF gene (a generous gift of Peter L. Davies) (30) into pBluescript (Stratagene). An EcoRI/NotI fragment was then blunt-end-ligated into the Hind111 site of the pSVOALA5' (27). pON249, a P-galactosidase expression vector under the control of the human cytomegalovirus promoter (31), was used to control for transfection efficiency.
Radioligand Binding-Neonatal rat hearts were excised, macroscopically freed from connective tissue, placed into ice-cold 1 mM KHCO,, and homogenized with a Tissumizer for 10 s at full speed and then twice for 20 s at 2/3 speed. The homogenate was washed twice at 40,000 X g for 30 min at 4 "C, and the final pellet was resuspended in binding buffer (50 mM Tris, 0.5 mM EDTA, pH 7.5) and rehomogenized for 10 s at full speed. [ 3 H ] P r a z~~i n binding was carried out for 45 min at 25 "C in 1 ml of binding buffer. The incubation was terminated by rapid vacuum filtration over Whatman GF/C filters which were washed twice with 10 ml of binding buffer. Nonspecific binding was defined as binding in the presence of 10 p~ phentolamine. Nonspecific binding was typically 10-15% of total binding at 0.3 nM [3H]prazosin. Protein content was determined by the method of Bradford (32) using bovine serum albumin as standard. Drug affinities at the cardiac a,-adrenergic receptor and receptor subtype distribution were assessed in competition binding experiments using -0.3 nM [3H]prazosin and pooled hearts of 18-25 neonatal rats (-300-500 pg of membrane protein per assay tube). The radioligand competition curves were analyzed by iterative nonlinear regression analysis to fit the experimental data to a mono-or biphasic sigmoid curve using the In Plot program (Graph PAD Software, San Diego, CA). The biphasic fit was accepted if it yielded statistically significant improvement in an F-test. From these curves, the apparent affinity (Ic50) at the high and low affinity sites and the percentage of sites in the high affinity state were calculated. ANF, atrial natriuretic factor; GTPyS, guanosine 5'-0-(thi0)triphosphate; G-proteins, guanine nucleotide binding proteins. conditions using six concentrations of ligand ranging from 0.1 to 1.6 nM. Affinity (Kd and number (Emx) of al-adrenergic receptors were determined from these saturation binding experiments with fitting of the untransformed data to a rectangular hyperbolic function (Fig. Phosphoinositide Hydrolysis-Myocardial cells were plated at a density of 3 X lo' cells/cm2 in 35-mm plates. Following a 24-h incubation in serum-containing medium, cells were washed and maintained in serum-free medium containing 5-30 pCi/ml [3H]inositol for 24 h. The cells were then rinsed in fresh serum-free medium and preincubated for 15 min with 2 CM DL-propranolol and 5-methylurapidil or (+)-niguldipine. 100 p M Phenylephrine and 10 mM LiCl were then added for 20 min, the medium was aspirated, the cells were harvested by scraping into 10% trichloroacetic acid and sonicated, and the cell homogenates were centrifuged for 10 min at 4 "C. Supernatants were neutralized by ether extraction and [3H]inositol phosphates were fractionated by anion exchange column chromatography as described (33).
Data Analysis-Results of multiple experiments are shown as mean f S.E. IC60 data from competition binding or inhibition experiments were converted to K, values using the Cheng and Prusoff (34)

RESULTS
Radioligand Binding Studies-To determine the relative composition of a-adrenergic subtypes in ventricular cells, radioligand binding studies were performed with a membrane fraction isolated from neonatal rat hearts. Saturation studies with [3H]prazosin detected 99 f 10 fmol (Kd=72 k 12 PM, n = 3 ) of a,-adrenoreceptors/mg of protein in neonatal rat heart membranes (Fig. U). In competition studies, the nonselective al-adrenergic agonist, phenylephrine, competed for [3H]prazosin binding with a steep monophasic curve. However, the ala-selective antagonists (+)-niguldipine, 5-methylurapidil, and oxymetazoline competed for [3H]prazosin binding in cardiac membranes from neonatal rats with shallow biphasic curves. ( Table I.) A relative distribution of approximately 32% ala-and 68% alB-adrenergic receptors was calculated from the ratio of high/low affinity sites for the subtype-selective compounds.
Phosphoinositide Hydrolysis-To determine the consequences of activation of specific a-adrenergic receptor subtypes, we examined inhibition of phenylephrine-stimulated phosphoinositide hydrolysis by al-adrenergic receptor antagonists. (+)-Niguldipine inhibited phenylephrine-stimulated inositol phosphate formation with high affinity (log Ki = -10.6) (Fig. 2). Decreasing the extracellular free Ca2+ concentration by addition of EGTA to -100 nM partially inhibited the response to phenylephrine; however, the K, for (+)-niguldipine was unchanged, suggesting that the Ca2+ channel blocking properties of niguldipine do not contribute to its potency in blocking phosphoinositide hydrolysis (data not shown). 5-Methylurapidil inhibited phosphoinositide hydrolysis with a log K; of -8.7. The K, values for (+)-niguldipineand 5-methylurapidil-mediated inhibition of phosphoinositide hydrolysis are similar to those anticipated for the high affinity @,,-adrenergic receptor. Phenylephrine also increased inositol  Table I.

TABLE I Affinity of adrenergic drugs for neonatal rat heart al-adrenoceptors
Data are the means f S.E. of values from three experiments in which 18-25 hearts were pooled. The competition curve for phenylephrine was monophasic. bis-and trisphosphate formation. Similar Ki values were determined for inhibition of inositol tris-, bis-, or monophosphate (data not shown). These data suggest that the increase in inositol monophosphate induced by phenylephrine results primarily from alA-adrenergic receptor-mediated polyphosphoinositide hydrolysis. Activation of an ANF Luciferase Fusion Vector via an ~I A -Receptor Subtype-Neonatal rat ventricular cells were transfected as described under "Materials and Methods" with the pANF638L luciferase reporter gene containing ANF 5"flanking sequences from -638 to +62 ligated upstream of the promoterless pSVOALA5' luciferase reporter gene (9). The 638 base pairs of ANF 5"flanking sequence is sufficient to confer the maximum level of al-adrenergic inducibility to the luciferase reporter gene in transient assays. In all experiments, transfection efficiency was monitored by co-transfection of the cytomegalovirus &galactosidase vector, and luciferase activity was normalized to ,&galactosidase activity. Following transfection, the cells were maintained in the absence or the presence of the 100 p M phenylephrine plus 2 FM DLpropranolol and either 10 p~ chlorethylclonidine or various concentrations of the alA-antagonists 5-methylurapidil or (+)niguldipine. 5-Methylurapidil and (+)-niguldipine decreased luciferase expression in these transfected cells in a concentration-dependent manner with a log Ki of -8. respectively (Fig. 3). Values are similar to the high affinity binding site observed in the radioligand binding studies in Table I and in previous studies of high affinity alA-adrenergic receptors in other cell types (18,35,36). Similar concentrations of 5-methylurapidil and (+)-niguldipine were required for inhibition of phosphoinositide hydrolysis ( Table 11). Treatment of cells with 10 p~ chlorethylclonidine had no effect on phenylephrine-stimulated activation of the ANF luciferase reporter gene (data not shown). al-Adrenergic stimulation does not induce luciferase expression in cells transfected with the constitutively expressed Rous sarcoma virus luciferase fusion gene or the promoterless pSVOALA5', demonstrating the specificity of adrenergic stimulation on the transcriptional activation of the ANF luciferase fusion gene (9).

5-Methylurapidil (+)-Niguldipine
Low affinity binding site -6. . Chlorethylclonidine (10 PM) had no significant effect on the phenylephrine-stimulated increase in cell size (data not shown). These findings correspond with the effect of the alA-antagonists on other markers of cultured ventricular cell growth (Table 11) and suggest that the alA-adrenergic receptor activates the morphologic features of the hypertrophic phenotype in a-adrenergically stimulated cultured ventricular myocytes.

DISCUSSION
Neonatal rat myocardium contains high and low affinity binding sites for the alA-adrenergic receptor-selective drugs oxymetazoline, 5-methylurapidil, and (+)-niguldipine. The drug affinities at the high and low affinity sites are in agreement with those previously described for a 1~-and alB-adrenergic receptors (18,35,36). Thus, ( Y~A -and als-adrenergic receptors coexist in rat neonatal ventricular myocardium in an approximately 30:70% ratio. This ratio is similar to that found previously in whole adult rat myocardium using the same ligand (18, 19), but the percent of alA-adrenergic receptors is somewhat lower than that reported in neonatal rat myocardium using ['251]BE 2254 as the ligand (37). The effect of stimulation of these receptor subtypes in myocardial cells is not well characterized. Therefore, the effect of alA-adrenergic receptor subtype antagonists on phosphoinositide hydrolysis, transcriptional activation of an ANF luciferase reporter gene, and cell size, three quantifiable markers of a-adrenergic-alA-Adrenergic Receptor-mediated Effects in Cultured Myocytes  H ) . Cells were then processed for immunocytofluorescent analysis using antibodies directed against a TrpE/MLC-2 fusion protein as described (24). ,!,eft panels were photographed at OX magnification; right panels at 63X. mediated cultured ventricular cell hypertrophy, were analyzed. Our data demonstrate that the alA-selective antagonists inhibited all three responses with high affinity (Table 11).
Previous classifications for al-adrenergic receptors have coupled the alB-adrenergic receptor to the formation of inositol phosphates and release of intracellular Ca"; whereas, the cY1A-adrenergiC receptor has been coupled to Ca2+ influx (17, 38). For example, formation of 1,4,5-inositol trisphosphate which has been implicated in the release of Ca2+ from intracellular stores was detected with stimulation of a l~adrenergic receptor in hepatocytes, but not with alA-adrenergic receptors in renal cells (39). More recent data, however, suggest that the response to a I A -and alB-adrenergic receptor stimulation may be dependent on the cellular context. For example, it has been shown that alA-adrenergiC receptor stimulation can elicit inositol monophosphate formation in renal cells (21, 39,40) and in primary cultures from rat brain (41). In renal cells, the alA-adrenergic receptor-stimulated inositol monophosphate formation was inhibited by removal of extracellular Ca2+ and was not associated with increases in inositol 1,4,5-trisphosphate (39) suggesting that the effect on inositol monophosphate formation was secondary to increases in intracellular Ca2+ and involved a different phosphoinositide hydrolysis pathway from that described for alB-adrenergic receptors. There is also recent data which demonstrate that in cultured ventricular myocytes, adrenergically induced polyphosphoinositide hydrolysis is inhibited by the cYIA-antagonist WB 4101 and not the irreversible alB-antagonist chlorethylclonidine (37). Our data similarly show that the a-adrenergic-mediated production of inositol trisphosphate in cardiac myocytes is inhibited by 5-methylurapidil and (+)niguldipine at concentrations that block the cm-adrenergic receptor, but not the alB-adrenergic receptor. In other studies: we have demonstrated that an antibody to the a-subunit * K. U. Knowlton of Gdll blocks phenylephrine-and GTPyS-stimulated inositol trisphosphate formation in membranes from neonatal ventricular myocytes. Taken together, these data suggest that a pharmacologically identifiable cY1A-adrenergic receptor subtype couples to the Gdll protein and stimulates phosphoinositide hydrolysis through activation of a polyphosphoinositide-specific phospholipase C in ventricular myocytes.
al-Adrenergic agonists initiate a program of hypertrophy in neonatal rat ventricular muscle cells that has molecular and morphologic characteristics similar to ventricular hypertrophy in the intact animal (7,8). The constellation of biochemical, genetic, and morphologic effects of a-adrenergic stimulation on cardiac ventricular muscle cells, and the previously mentioned diversity of al-adrenergic receptor subtypes, suggests the possibility that these effects may be the result of the simultaneous activation of multiple a,-adrenergic receptor subtypes, and that the phenotypic effects represent an integrated response to the activation of a variety of signaling pathways. However, the results of the present study suggest that not only are proximal signaling mechanisms in adrenergic receptor-mediated cultured ventricular cell hypertrophy under the control of the alA-adrenergic receptor, but that the a,,-adrenergic receptor also functions like a growth factor receptor in neonatal cardiac myocytes, stimulating long term genetic and morphologic features of cultured myocardial cell hypertrophy.
A variety of cell types and conditions have been used to evaluate the response to a-adrenergic stimulation that occurs within minutes of ligand binding (17,21,22,(38)(39)(40)(41). However, little is known regarding the pathways which mediate the long term effects of a,-adrenergic stimulation on transcriptional activation that contribute to myocardial cell growth. The a,-, cyz-, and @-adrenergic receptor subtypes are each coupled to distinct G-proteins which act to transduce Gprotein-specific responses, such as activation or inhibition of adenylylcyclase, or activation of phospholipase C. The aminoterminal portion of the third intracellular domain of the adrenergic receptor plays a major role in determining the Gprotein specificity of a given adrenergic receptor. B-Adrenergic receptors in which the third intracytoplasmic domain is replaced by the third intracytoplasmic domain from the al" adrenergic receptor can activate phosphoinositide hydrolysis in COS7 cells (22,42). In addition, a single amino acid substitution in the third intracellular domain of the alMadrenergic receptor renders the receptor constitutively active as assessed by phosphoinositide hydrolysis (43). Transfection of mutated alB-adrenergic receptor expression vectors into Rat-1 and NIH 3T3 fibroblasts induces proliferation even in the absence of catecholamines (44). These previously reported effects of mutations in the alB-adrenergic receptor in noncardiac cells and the findings of the present manuscript demonstrating the effects of stimulation of the aIA-adrenergic receptor in cardiac myocytes suggest that the adrenergically mediated model of myocardial cell hypertrophy can be utilized to precisely characterize the effects of mutations in an ~I Aadrenergic receptor. There is evidence by expression cloning for the presence of at least three distinct al-adrenergic receptor subtypes (45,46), but the gene corresponding to the pharmacologically defined rat cardiac alA-adrenergic receptor has not been isolated (46). Microinjection and transfection of mutant ala-adrenergic receptor expression vectors into CUItured ventricular myocytes will allow quantitative assessment of the effects of these mutations on ANF gene expression and myocardial cell size.
Recent studies have documented that activation of another G-protein-coupled receptor, endothelin-1, will also activate the hypertrophic response of cultured ventricular muscle cells, in a manner analogous, both quantitatively and qualitatively, to the effects of alA-adrenergic receptor stimulation. The endothelin-1 receptor is linked to phosphoinositide hydrolysis and the up-regulation of both the ANF and MLC-2 genes. Furthermore, similar cis regulatory elements within the ANF and MLC-2 promoters appear to be required for inducible expression by either endothelin or a-adrenergic agonists (29,47). The addition of a-adrenergic agonists and endothelin-1 does not have an additive effect on cultured ventricular muscle cells. Thus, it appears that these two distinct stimuli converge on similar signaling pathways for the activation of genetic markers of the hypertrophic response. Identification of the specific receptors which activate these quantitative and qualitative features of neonatal ventricular cell hypertrophy will now allow genetic manipulation of each receptor macromolecule to precisely characterize its potential interaction with other proximal signaling molecules involved in the hypertrophic response.
The current studies, therefore, demonstrate that the pharmacologically identified a,,-adrenergic receptor activates biochemical, genetic, and morphologic markers of ventricular muscle cell hypertrophy. The precise role of this G-proteincoupled receptor as a myocardial growth factor receptor, and its interaction with other proximal signaling molecules such as the G-proteins, G,, and Ras, can now be assessed using cotransfection and microinjection in cultured ventricular myocardial cells, and in transgenic mice (48). These studies are currently in progress and should shed further light on the aIAadrenergic receptor-dependent signaling pathways which lead to the activation of ventricular muscle cell hypertrophy.