©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Adrenergic Receptor Subtype mRNAs Are Differentially Regulated by -Adrenergic and Other Hypertrophic Stimuli in Cardiac Myocytes in Culture and In Vivo
REPRESSION OF alpha AND alpha BUT INDUCTION OF alpha(*)

D. Gregg Rokosh Alexandre F. R. Stewart Kevin C. Chang Beth A. Bailey Joel S. Karliner S. Albert Camacho (1) Carlin S. Long Paul C. Simpson (§)

From the Cardiology Division and Research Service, Veterans Affairs Medical Center, San Francisco, California 94121, the Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143, and the Department of Medicine (Cardiology), San Francisco General Hospital, San Francisco, California 94110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The three cloned alpha(1)-adrenergic receptor (AR) subtypes, alpha, alpha, and alpha, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three alpha(1)-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the alpha(1)-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12-72 h). alpha and alpha mRNA levels were repressed by NE, whereas alpha mRNA was induced. Changes in mRNA levels were mediated by an alpha(1)-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [^3H]prazosin binding. alpha(1)-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2alpha, and phorbol 12-myristate 13-acetate, also induced alpha mRNA and repressed alpha mRNA. In myocytes from hearts with pressure overload hypertrophy, alpha(1) mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among alpha(1)-AR subtypes expressed natively in the same cell. Transcriptional induction of the alpha-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.


INTRODUCTION

The natural catecholamines norepinephrine (NE) (^1)and epinephrine activate adrenergic receptors (ARs) in three families, alpha(1), alpha(2), and beta. Multiple subtypes have been cloned within each family: three alpha(1)-ARs (B, C, and D), (^2)three alpha(2)-ARs (A, B, and C; also called C10, C2, and C4, respectively), and three beta-ARs (1, 2, and 3)(1) . The reason(s) for conservation of multiple subtypes remain uncertain, since all subtypes in each family couple preferentially to the same effector when overexpressed, alpha(1)-ARs to activation of phospholipase C (PLC), alpha(2)-ARs to inhibition of adenylyl cyclase, and beta-ARs to activation of adenylyl cyclase(1) .

An intriguing difference among beta-AR and alpha(2)-AR subtypes has been suggested recently, in the regulation of receptor levels during prolonged agonist exposure. Levels of the beta(3)-AR (2, 3, 4) and the alpha-AR (5, 6) are not down-regulated during long term agonist exposure, at least in some cells, in contrast with down-regulation of beta(1)- and beta(2)-ARs, and alpha- and alpha-ARs. Down-regulation of receptor expression is thought to be a major determinant of desensitization when catecholamine exposure is prolonged(7) . Conversely, a receptor that is induced by agonist might be adapted to mediate catecholamine responses when sympathetic activity is increased chronically, a condition that occurs frequently in the intact organism.

We have been studying a physiological response that develops over long periods of catecholamine exposure, alpha(1)-adrenergic induction of hypertrophic growth and gene transcription in primary cultures of neonatal rat cardiac myocytes(8, 9, 10, 11, 12, 13) . We have found recently that the cardiac myocytes express the mRNAs for all three cloned alpha(1)-AR subtypes, the alpha, the alpha, and the alpha, whereas cardiac fibroblasts do not express any alpha(1)-AR(14) . This system thus provides the opportunity to study regulation of all three alpha(1)-AR subtypes expressed natively in the same cell. Most prior studies of chronic agonist regulation of the beta(3)- and alpha-ARs have been in transfected cells overexpressing these subtypes(3, 4, 5, 6) , where transcriptional regulation of receptor expression would not occur.

In the present study, we report that the alpha(1)-AR subtype mRNAs respond in distinctly different ways during chronic exposure to NE. The mRNAs encoding the alpha and the alpha were repressed, whereas alpha mRNA was induced. These mRNA changes were mediated through an alpha(1)-AR, were likely transcriptional in origin, and were followed by changes in receptor protein by radioligand binding. alpha(1)-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists, endothelin-1 (ET-1)(15) , PGF2alpha(16) , and phorbol 12-myristate 13-acetate (PMA)(17) , also induced alpha mRNA and repressed alpha mRNA. Changes identical to those produced by NE in culture were seen in myocytes isolated from hearts with hypertrophy produced by abdominal aortic banding. These results provide the first example of a difference in regulation among alpha(1)-AR subtypes expressed natively in the same cell. Transcriptional induction of the alpha-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.


EXPERIMENTAL PROCEDURES

Cell Culture

Myocytes were isolated from the day-old rat heart using trypsin and were seeded at single-cell density in minimal essential medium with 5% calf serum (Hyclone) and 0.1 mM bromodeoxyuridine to inhibit fibroblast proliferation(18, 19) . After 26-28 h, cells were washed and incubated in serum-free minimal essential medium containing 10 µg/ml bovine transferrin (Hyclone), 10 µg/ml porcine insulin (Lily), 1.0 mg/ml bovine serum albumin (Armour), 0.1 mM bromodeoxyuridine, and 100 µM ascorbic acid as an antioxidant. Drugs or their vehicle were then added, and the cultures were incubated for increasing times at 37 °C.

RNase Protection Assay

Total RNA (20) was used in RNase protection assay with probes specific for each of the three rat alpha(1)-AR mRNAs, alpha, alpha, and alpha, and with probes for glyceraldehyde-phosphate dehydrogenase (GAPDH) and beta-actin, as described and validated previously(14, 21) . The rat beta-actin probe (188 bases; Ambion) protected a fragment of 126 bases (coding sequence nucleotides 771-896)(22) . In most experiments, the autoradiographic bands for each mRNA were scanned at 600 dpi and quantified as arbitrary density units/µg of total RNA (30-50 µg of RNA in each assay) (Scan Analysis, Biosoft). In the pressure overload experiments, bands were quantified by phosphor imaging (Bio-Rad GS-363 Molecular Imager). In all cases, signals were corrected for background and were within the linear range of the assay.

[^3H]Prazosin Binding

A total membrane fraction (100,000 times g pellet of a myocyte homogenate) was used for [^3H]prazosin binding, with 10 µM phentolamine to define nonspecific binding, as described previously (14) . In competition binding, membranes in assay buffer (1.0 ml final volume) were incubated at 30 °C in a shaking water bath with 22 concentrations of the alpha(1)-adrenergic antagonist 5-methylurapidil (5MU) (0.01-100 nM) for 5 min before the addition of 0.25 nM [7-methoxy-^3H]prazosin (72 Ci/mmol, DuPont NEN). A [^3H]prazosin K(D) of 0.103 ± 0.013 nM (n = 5) was used for competition binding analyses with the iterative curve-fitting program EBDA/LIGAND (23) ; NE treatment for 48 h had no effect on the [^3H]prazosin K(D) by saturation binding (data not shown). Differences between competition binding curves were determined by two-factor repeated measures analysis of variance.

Phosphatidylinositol Hydrolysis

Myocytes in triplicate 35-mm dishes were radiolabeled with 5 µCi/ml myo-[2-^3H]inositol (4 Ci/mmol, Amersham Corp.) for 48 h, rinsed, and treated with vehicle or six concentrations of NE (1 nM to 100 µM) in the presence of 10 mM LiCl. After 10 min at 37 °C, cellular [^3H]inositol phosphates (IPs) were extracted with trichloroacetic acid and separated by anion-exchange chromatography, as described previously(24) .

Cultured Myocyte Growth

Myocytes in triplicate 35-mm dishes were incubated with vehicle or four concentrations of NE (20 nM to 20 µM) for 72 h in the presence of 0.1 µCi/ml L-[U-^14C]phenylalanine (498 mCi/mmol, DuPont NEN). Myocyte size was quantified from the content of radiolabeled protein per dish. Cell numbers are unchanged over time in these cultures, and >90% of the cells are myocytes(8, 9) .

Aortic Banding and Isolation of Adult Cardiac Myocytes

Adult male Sprague-Dawley rats (200-220 gm) were anesthetized with pentobarbital, and a 0.31-mm internal diameter Weck hemoclip was placed around the suprarenal abdominal aorta through a left flank incision. For sham controls, the clip was not closed. After 10-12 weeks, cardiac myocytes were isolated by retrograde perfusion of the heart with collagenase, as described previously(14) . Myocyte yield was 10.9 ± 0.2 times 10^6/heart for banded rats (n = 4) and 10.1 ± 0.6 times 10^6/heart for shams (n = 5), and >90% of cells were rod-shaped. An aliquot of the isolated myocytes was used to measure cell volume on a Coulter Multisizer, and the rest were used to prepare RNA(20) .

Materials

(-)-NE HCl, human/porcine ET-1, PMA, and (-)-timolol maleate were from Sigma; prazosin HCl and 5MU, from Research Biochemicals Inc. PGF2alpha was from Caymen Chemicals, [5,6-^3H]uridine (37 Ci/mmol) was from DuPont NEN, and actinomycin D was from Calbiochem. Stock solutions were in 1 mM HCl, or in Me(2)SO for prazosin, actinomycin D, PGF2alpha, and PMA (final Me(2)SO leq 0.01 volume %).

Data Analysis

Results are presented as the mean ± SE from the number of experiments indicated. Treated/control ratios were tested for deviation from unity by calculation of confidence limits. Mean values were compared by the paired, two-sided Student's t test.


RESULTS

In this neonatal rat cardiac myocyte culture model, alpha(1)-AR-induced growth and gene expression are half-maximum at 12-18 h and maximum at 24-48 h of catecholamine exposure(9, 10) ; and these responses require continuous receptor occupancy by agonist(25, 26, 27) . Thus, continuous alpha(1)-AR signaling appears to occur over long times, seemingly counter to the well established concept of AR desensitization by agonist(7) .

To determine if prolonged catecholamine exposure produced atypical regulation of one or more of the three alpha(1)-AR subtypes in the myocytes, the alpha(1)-AR mRNAs were quantified by RNase protection after treatment with NE. A dose of NE was used (2 µM) that is maximum for myocyte growth and gene transcription(9, 10) . After 24 h of NE exposure, alpha and alpha mRNA levels were reduced to about 26 and 42%, respectively, of those in control myocytes treated concurrently with vehicle (Fig. 1). Repression of alpha and alpha mRNAs was also evident when normalized to the levels at time 0, when NE was added (19 ± 1% and 14 ± 1% for alpha and alpha, respectively, n = 5, p < 0.05). (^3)In direct contrast to the decrease of alpha and alpha mRNAs, NE increased the abundance of alpha mRNA, by over 3-fold (Fig. 1). Induction of alpha mRNA was also observed when normalized to the level at time zero (4.4 ± 1.2-fold, n = 5, p < 0.05). As a control, NE had no significant effect on the mRNA for the ``housekeeping'' gene GAPDH (at 24 h, 1.16 ± 0.10-fold versus vehicle, 1.10 ± 0.08-fold versus time 0, n = 3, p = not significant). The nonselective alpha(1)-AR antagonist prazosin inhibited regulation of the alpha(1)-AR mRNAs by NE, whereas the beta-AR antagonist timolol did not (Fig. 1). Thus activation of an alpha(1)-AR induced alpha mRNA and repressed alpha and alpha mRNAs.


Figure 1: NE induces alpha mRNA and represses alpha and alpha mRNAs in cardiac myocytes through an alpha(1)-AR. Cultured cardiac myocytes were treated with 2 µM NE or vehicle control in the absence or presence of the alpha(1)-AR antagonist prazosin (Pzn) (2 µM) or the beta-AR antagonist timolol (Tim) (2 µM), added 30 min before NE. After 24 h, the alpha(1)-AR subtype mRNAs were quantified by RNase protection. Values are the mean ± S.E. treated/control ratios of mRNA band density/µg of total RNA for the number of experiments indicated below the bars, with seven 100-mm dishes used for each group in each experiment. Prazosin or timolol alone had no significant effect on any mRNA (data not shown). *, p < 0.05 versus vehicle control.



As shown in Fig. 2, the effects of NE on each mRNA were sustained for up to 72 h of continuous NE exposure. The effects were detectable at the earliest time studied (2 h; alpha 1.4-fold times control, alpha 76% of control, and alpha 37% of control; means from two experiments), were submaximum at 12 h (two experiments as in Fig. 3), and were maximum at 24 h (four experiments as in Fig. 2).


Figure 2: NE regulation of alpha(1)-AR mRNAs is sustained over 72 h. Cultured myocyte RNA was harvested for RNase protection assay at the time of 2 µM NE addition (0 h), and at 24, 48, and 72 h after the addition of NE (+) or vehicle(-). Protected fragments of the following sizes (bases) are shown: alpha, 432; alpha, 315; alpha, 217; and GAPDH, 316(21) . Yeast transfer RNA (tRNA) was a control for nonspecific hybridization, and GAPDH was a control for input RNA. The same results were observed in three additional experiments, and the numbers were similar to those quantified in Fig. 1. At 72 h, the level of alpha mRNA in cells treated with NE alone was 25% of the vehicle control, whereas it was higher, 70% of control, with NE in the presence of timolol (2 µM), suggesting a beta-AR contribution to persistent alpha mRNA down-regulation (mean from 3-4 experiments).




Figure 3: NE has no effect on alpha(1)-AR mRNA degradation. Cultured cardiac myocytes were treated with actinomycin D (+Act D) (0.05 µg/ml) or with its vehicle (-) in minimal essential medium with 1% calf serum for 3 h, and then 2 µM NE (+NE) or its vehicle(-) were added at time 0 (0 h). RNA was harvested at the times indicated (-3 to 12 h), and the alpha(1)-AR mRNAs and GAPDH mRNA were assayed by RNase protection. Protected fragments are as in Fig. 2, except a longer alpha riboprobe, 474 bases, was used to protect 446 bases of alpha mRNA (nucleotides 1752-2197(52) ). The same results were obtained in an additional experiment, and receptor mRNA degradation half-lives (see ``Results'') were estimated from semilogarithmic plots of mRNA levels versus time in the presence of actinomycin D.



To test if the NE-induced changes in mRNA levels were transcriptional in origin, the myocytes were incubated with actinomycin D, at a concentration (0.05 µg/ml) that inhibited transcription by >95% within 3 h, as assayed by [^3H]uridine incorporation into total RNA, but had no effect on cell viability over 12 h (data not shown). As shown in Fig. 3, all three alpha(1)-AR mRNAs disappeared rapidly in the presence of actinomycin D, with apparent degradation half-lives of 2.5, 3, and 1 h for alpha, alpha, and alpha mRNAs, respectively (Fig. 3, lanes 1-3 and 5). Treatment with NE when transcription was inhibited by actinomycin D had no effect on the abundance of any alpha(1)-AR mRNA (Fig. 3, lanes 3-6). In the absence of actinomycin D, in contrast, the characteristic effects of NE on all three mRNAs were observed over the same time interval (12 h; Fig. 3, lanes 7 and 8). These results indicated that NE did not alter alpha(1)-AR mRNA stability and suggested that alpha(1)-AR stimulation regulated transcription of the alpha(1)-AR genes.

Radioligand binding was used to test whether the changes in receptor subtype mRNAs were accompanied by parallel changes in receptor proteins. In competition binding assays with [^3H]prazosin, the cloned rat alpha-AR has much higher affinity for the antagonist 5MU (4 nM) (28) than does the rat alpha-AR (122 ± 4 nM) (28, 29) or the rat alpha-AR (140 ± 120 nM) (28, 29, 30, 31, 32) . In the cultured cardiac myocytes, competition binding with [^3H]prazosin distinguished an alpha(1)-AR population with high affinity for 5MU (4.4-7.6 nM) and a population with low affinity (261-277 nM) (Table 1). The high affinity sites were assumed to reflect the alpha-AR, and the low affinity sites were assumed to reflect the alpha-AR and/or the alpha-AR. (^4)Exposure to NE for 72 h decreased the number of low affinity sites to 60% of control, consistent with down-regulation of the alpha and/or the alpha (Table 1). In contrast, NE exposure for 72 h doubled the number of high affinity sites, consistent with up-regulation of the alpha-AR (Table 1) and in agreement with the 3-fold increase in alpha mRNA (Fig. 1). Thus in the cardiac myocytes exposed chronically to NE, the alpha-AR became the predominant receptor, increasing from 26 to 55% of total alpha(1)-ARs (Table 1). It was noteworthy that total receptor number did not change, despite the significant shift in subtype proportions (Table 1).



PLC activation was used to test for desensitization of alpha(1)-AR signaling. alpha(1)-AR coupling to PLC in cultured neonatal rat cardiac myocytes is through an alpha(1)-AR with high affinity for 5MU (2 nM)(33) , probably the alpha (see above for 5MU affinities of cloned alpha(1)-ARs). In myocytes exposed to 2 µM NE for 48 h, the EC for NE-stimulated total [^3H]IP production was unchanged (0.7 µM for NE-treated cells versus 0.9 µM for control cells, mean of four experiments). Fractions corresponding to IP(1), IP(2), and IP(3) were increased equally in NE-treated and control cells (data not shown), and maximum total [^3H]IP responses were not different (3.6-fold for NE-treated cells versus 3.3-fold for controls). Thus there was no desensitization of alpha(1)-AR-mediated PLC activation with 72 h of NE exposure, consistent with prior studies in cultured myocytes (34, 35) and with unimpaired alpha-AR signaling. It was not possible to test for desensitization of alpha and/or alpha signaling, since biochemical responses coupled to these alpha(1)-ARs in myocytes have not been identified conclusively.

There was also no desensitization of a physiological response to chronic alpha(1)-AR activation, NE-induced myocyte growth. In myocytes pretreated with 2 µM NE for 72 h, the EC for NE-stimulated protein accumulation over the subsequent 72 h was the same as in control myocytes (0.8 ± 0.3 µM for NE-treated cells versus 0.7 ± 0.3 µM for controls, n = 5, p = not significant).

To test if induction and repression of the alpha(1)-AR subtype mRNAs required alpha(1)-AR occupancy, mRNA levels were measured after treatment of the myocytes with three other hypertrophic growth factors, ET-1(15) , PGF2alpha(16) , and PMA(17) . As shown in Fig. 4, ET-1, PGF2alpha, and PMA were all similar to NE, inducing alpha mRNA and repressing alpha mRNA. Interestingly, unlike NE, PGF2alpha tended to induce alpha mRNA, and PMA had no effect on alpha (Fig. 4).


Figure 4: Hypertrophic stimuli induce alpha mRNA and repress alpha mRNA in culture and in vivo. Cultured neonatal rat cardiac myocytes were treated for 24 h with 2 µM NE, 100 nM ET-1, 10 µM PGF2alpha, 100 nM PMA, or their vehicle controls. Adult cardiac myocytes were isolated from the intact rat heart after 10-12 weeks of aortic banding. Total RNA was prepared and the alpha(1)-AR subtype mRNAs were quantified by RNase protection. For the growth factors in culture, values are the mean ± S.E. treated/control ratios for three separate experiments (five for PMA). For aortic banding, values are the mean banded/sham ratios for four banded rats and five shams; statistical analyses were done on the absolute phosphor imaging values. *, p < 0.05 versus vehicle control.



The pattern of alpha(1)-AR mRNA regulation produced by NE in culture was also observed with a pressure overload stimulus for hypertrophy in the intact animal. Myocytes were isolated from the adult rat heart after 10-12 weeks of abdominal aortic banding. Banding stimulated myocyte hypertrophy, increasing the mean volume of isolated myocytes by 20%, from 36,000 ± 300 µm^3/myocyte with sham operation to 43,000 ± 900 µm^3 with banding (n = 4-5 hearts, p < 0.001). As shown in Fig. 4, aortic banding increased alpha mRNA level by almost 3-fold and decreased the levels of alpha and alpha significantly, to 71 and 56% of sham, respectively. As a control, banding did not change the level of myocyte beta-actin mRNA (1.14-fold versus sham).


DISCUSSION

These results provide the first example of a difference in regulation among alpha(1)-AR subtypes expressed natively in the same cell. The alpha- and alpha-AR mRNAs were repressed by chronic alpha(1)-AR stimulation of cultured cardiac myocytes, whereas alpha mRNA was induced, and there were coordinate changes in alpha(1)-AR binding activity. A similar pattern of mRNA induction and repression was seen with other hypertrophic agonists in culture, ET-1, PGF2alpha, and PMA, and with a pressure overload stimulus for hypertrophy in the intact animal, aortic banding. Our results differ from overexpression studies in rat 1 fibroblasts, where all three alpha(1)-AR subtypes are down-regulated equally by agonist(36) , and thus emphasize the importance of transcriptional control of native alpha(1)-AR expression.

The observed differences in subtype regulation might be physiologically important. Induction of the alpha-AR is a potential mechanism for sustained signaling during hypertrophic growth, a chronic physiological response to catecholamines in cardiac myocytes. alpha(1)-AR-stimulated growth and transcription in cultured myocytes requires sustained alpha(1)-AR signaling over long times, since the trophic effects decay whenever agonist is removed or antagonist is added(25, 26, 27) . Consistent with these earlier results, we found here that exposure of cultured myocytes to NE for 3 days did not desensitize alpha(1)-AR-stimulated PLC activation or growth. Both responses have been attributed to an alpha(1)-AR with alpha-AR-like affinity for 5MU(33) , and it thus can be proposed that the responses are sustained because of alpha-AR induction. Enhanced sensitivity of PLC activation or growth was not observed with alpha-AR up-regulation. A possible explanation can be found in recent overexpression studies, where increases in alpha(1)-AR number in the 2-3-fold range, the increase in alpha-ARs we found in the present study, do not change PLC activation significantly(37) , possibly because G(q)alpha/Galpha are concurrently down-regulated(36) . Thus alpha-AR induction might sustain PLC activation and growth despite down-regulation of G proteins or other changes that could desensitize the responses.

Induction of alpha mRNA and repression of alpha were observed with several hypertrophic agonists in culture and with pressure overload in vivo, suggesting that these mRNA changes could be one feature of a specific hypertrophic transcriptional program, analogous to the induction in hypertrophy of genes expressed preferentially during fetal cardiac development, such as the contractile protein isogenes, beta-myosin heavy chain, and skeletal alpha-actin (for a review see (38) ). A transcription factor has been identified in cardiac myocytes, transcriptional enhancer factor-(TEF-1), that is involved somehow in alpha(1)-adrenergic activation of the beta-myosin heavy chain and skeletal alpha-actin promoters(11, 12, 13) . The rat alpha promoter, the only alpha(1)-AR promoter studied in detail so far, contains a potential TEF-1 binding site(39) . Thus it will be interesting to see if the alpha-AR is expressed preferentially during fetal cardiac development and if TEF-1 plays a role in alpha(1)-AR gene transcription. It will also be interesting to test whether alpha-AR induction is a required mechanism for sustaining cardiac growth induced by various hypertrophic stimuli.

The mechanisms for transcriptional control of alpha(1)-AR genes are likely to vary in different cell types. Expression of alpha(1)-AR subtype mRNAs is tissue- and cell-specific(14, 21) , and regulation of alpha(1)-AR mRNAs by agonist is also different in different cells. For example, in cultured smooth muscle cells, alpha mRNA is reduced minimally (40) or not at all (41) with prolonged catecholamine exposure, in contrast to the marked repression of alpha mRNA in cardiac myocytes observed in this study.

Even in myocytes, transcriptional controls might not be identical for all hypertrophic agonists, since PMA and PGF2alpha differed from NE in their failure to repress alpha mRNA. In this regard, it is notable that the pattern of alpha mRNA induction and alpha and alpha mRNA repression was identical with NE treatment in culture and pressure overload in vivo. We did not test whether NE was the proximate stimulus for the mRNA changes with aortic banding, but sympathetic activity is increased in animals and humans with hypertrophy (for examples see (42, 43, 44, 45) ).

In the transgenic mouse heart, overexpression of an activated alpha-AR induces cardiac myocyte hypertrophy(46) , possibly in conflict with the idea that the alpha-AR transduces growth in myocytes under native conditions(33) . On the other hand, this activated alpha-AR couples efficiently to PLC activation when overexpressed in cell lines(47, 48) , and the same appears to be true when it is overexpressed in myocytes, as judged by increased diacylglycerol accumulation(46) . Thus the activated alpha-AR might produce hypertrophy simply because it is able to stimulate PLC in myocytes. In addition, our results here suggest that stimulation of PLC by the activated alpha could induce the native alpha-AR in the transgenic hearts, and thus the native alpha might mediate the hypertrophic response. It will be important to test whether the alpha has some structural property that is required for growth, in addition or alternate to PLC activation.


FOOTNOTES

*
This work was supported by grants from the National Institutes of Health and the Department of Veterans Affairs Research Service and by research fellowships from the Heart and Stroke Foundation of Canada (DGR) and the American Heart Association, California Affiliate (AFRS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: VAMC 111-C-8, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-221-4810 (ext. 3200); Fax: 415-750-6950.

(^1)
The abbreviations used are: NE, norepinephrine; AR, adrenergic receptor; ET-1, endothelin-1; GAPDH, glyceraldehyde-phosphate dehydrogenase; IP, inositol phosphate; PLC, phospholipase C; PMA, phorbol myristate acetate; 5MU, 5-methylurapidil.

(^2)
It has been suggested that the alpha be renamed the alpha(49) . For clarity, we continue to use the name alpha here, since the designation alpha (or alpha) has also been applied to the alpha in many reports(49) .

(^3)
As shown in Fig. 2(lane 2 versus lane 1), the level of alpha mRNA was markedly lower in the absence of serum; thus down-regulation of alpha mRNA by NE was greater when normalized to time zero than to the vehicle control.

(^4)
Receptor inactivation with chloroethylclonidine (28, 50, 51) did not resolve a more sensitive alpha-AR and a less sensitive alpha-AR in the population of alpha(1)-ARs with low affinity for 5MU (data not shown).


ACKNOWLEDGEMENTS

We thank Marietta Paningbatan for excellent technical support, Marc G. Caron and Robert J. Lefkowitz for the rat alpha cDNA, and Robert M. Graham for the rat alpha cDNA.


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