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Agonist-directed sorting of β2-adrenergic receptor signaling and its therapeutic implication in heart
http://www.100md.com 《河南医科大学学报》 2000年第1期
     作者:ZHANG Shengjun XIAO Ruiping ZHOU Yingying Meike Kuschel XIA Xianmin Richard A.Bond Olga V.Fedorova Alex Y.Bagrov C.Wiuiam Balke Edward G.Lakatta CHEN Heping

    单位:Laboratory of Cardiovascular Science ,Gerontology Research Center,NIA,NIH,Baltimore,MD 21224

    关键词:adrenergic;beta;receptor;agonists;G;protein;toxins;cell;contra-ctihty▲

    河南医科大学学报000110ZHANG Shengjun,XIAO Ruiping,ZHOU Yingying,Meike Kuschel,XIA Xianmin,Richard A.Bond ,Olga V.Fedorova,Alex Y.Bagrov,C.Wiuiam Balke,Edward G.Lakatta,CHEN Heping
, 百拇医药
    (Laboratory of Cardiovascular Science ,Gerontology Research Center,NIA,NIH,Baltimore,MD 21224)

    Abstract:Cardiac β2-adrenergic receptor (AR) dually couples to Gs and to pertussis toxin(PTX)-sensitive Gi proteins,resulting in opposing effects on cell contractility.Here we show that the coupling preference of β2AR to the bifurcated signaling pathways,tested over wide ranges of agonist concentrations and receptor densities,is determined by agonist entities.Most β2AR agonists including zinterol,procaterol or salbutamol activate concurrent Gs and Gi signaling in both rat and mouse ventricular myocytes,as evidenced by a marked potentiating effect of PTX on contractile responses.Fenoterol,however ,sorts the β2AR primarily to Gs signaling,as evidenced by the lack of effect of PTX.Similar results were observed in transgenic mice overexpressing human β2AR.In cells from failing rat hearts where the contractile response to zinterol was severely depressed due to an up-regulation of Gi,fenoterol,which activates Gs bypassing the Gi signaling pathway,rescued the diminished β2AR response.Thus,agonist-directed sorting of receptor stimulus affords a novel therapeutic strategy to enhance desired “signaling-specific”effects while avoiding adverse side effects of therapeutic agents.
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    Key words:adrenergic beta receptor agonists;G protein; toxins;cell contra-ctihty▲

    Promiscuous coupling of a G-protein coupled receptor to more than one class of G proteins has recently been demonstrated in artificial systems,including lipid vesicles and transfected cell lines[1,2],and in native tissues and cells[3~5].A fundamental question arisen is how the receptor stimulus is sorted in the “one receptor,multiple G protein”signaling system.In the simplest“twostate”receptor model[6,7],ligands of a given receptor,either synthetic drugs of endogenous hormones or neurotransmitters,ought to activate all post-receptor pathways because a receptor has only one active conformational state.In contrast,the receptor“trafficking”hypothesis[8] predicts that ligands may promote the receptor at multiple active states,each having distinct G protein selectivity,and thereby directs the receptor stimulus to different signaling cascades.Although supporting evidence has emerged form studies of surrogated receptors in transfected cell lines[2],agonist-directed“trafficking”of receptor stimulus has not been demonstrated for any native receptor system,and its physiologic and pathophysiologic relevance is unknown.
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    In mammalian heart ,at least two β-adrenergic receptor(βAR)subtypes,β1AR and β2AR,coexist and stimulation of these prototypic G protein-coupled receptors by catecholamines serves as a main regulatory mechanism of cardiac performance.Our recent studies have shown that β2AR,unlike β1AR,dually couples to Gs and to pertussis toxin(PTX)-sensitive Gi proteins ,resulting in opposing effects on cell contractility[3,4].Using this unique“one receptor,two G protein”system
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    identified in intact heart cells,we screened an array of β2AR agonists——including fenoterol,zinterol,salbutamol and procaterol—to determine whether the β2AR-coupled Gs and Gi signaling cascades can be selectively activated,and if so,what are the physiological and therapeutic implications of the agonist-directed sorting of β2-adrenergic stimulation in heart.

    The cell contractile response and its PTX-sensitivity[9]were measured to index the overall output of the Gs(in PTX-treated cells) or combined Gs-Gi signaling (in non-PTX treated cells).The specificity of these β2AR agonists was verified by using selective β1AR and β2AR subtype antagonists,CGP 20712A(CGP) and ICI 118 551 (ICI),respectively[10].The β1AR selective antagonist CGP (10-8M)completely abolished the positive inotropic effect of a β1AR agonist norepinephrine (10-7M)in the presence of an α1AR blocker,prazosin(10-6M)(Figs.1A&G).In contrast,the
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    equipotent contractile response to the aforementioned β2AR agonists could not be reversed by CGP(Figs.1B&G),but were fully antagonized by the β2AR antagonist ICI(10-7M)(Figs.1C-F).

    As shown in Fig.2,salbutamol,procaterol and zinterol enhanced the contraction amplitude of rat heart cells in a dose-dependent manner,with a maximal response of ~200% of control(Figs.2A-C).PTX pretreatment,to inhibit Gi protein function,potentiated the contractile response and increased the maximal response to~300% of control(Figs.2A-C).A similar potentiating effect of PTX was observed for β2AR stimulation induced by terbutaline,celenbuterol and epinephrine or isoproterenol(ISO)plus the β1AR blocker CGP(data not shown).These results are in agreement with the previous notion that agonist-stimulated β2AR can interact with both Gi and Gs[3,4,11],and thereby provide the basis for testing receptor trafficking in the physiological context.
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    In sharp contrast to the β2AR agonists tested above,fenoterol,another

    selective β2AR agonist[12],induced a maximal contractile response of~300% of control in the absence of PTX,i.e.,it was twice as efficacious as the other β2AR agonists under the present experimental conditions (Fig.2D).More importantly,β2AR stimulation by fenoterol appeared to be qualitatively different from that induced
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    by the other β2AR agonists,because PTX had no significant effect on its contractile response over a wide range of concentrations (10-8M to 10-4M)(Fig.2D).These results suggest that fenoterol selectively activates the β2AR-coupled Gs signaling pathway,whereas the other β2AR agonists tested recruit concurrent Gs and

    Gi-mediated signaling pathways.
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    To determine whether the agonist-dependent selective interaction of β2AR and G proteins is present in other species,we next examined β2AR stimulation in mouse cardiac myocytes.Remarkably,β2AR stimulation by zinterol (Fig.3 A) or by ISO (10-6M)in the presence of the β1AR selective blocker CGP (10-8M)( data not shown) failed to elicit a significant contractile response.While this might suggest that murine cardiac β2AR is nonfunctional in terms of contractile response,PTX treatment unmasked a marked dose-dependent positive inotropic effect of zinterol (Fig.3A),converting the apparently quiescent β2AR agonism into a full β2AR agonism.These observations qualitatively agree with the data observed in rat cardiomyocytes,except that the murine β2AR-Gi signaling totally negates the Gs-mediated positive inotropic effect.Strikingly,β2AR stimulation by fenoterol in non-PTX treated cells augmented cell contraction to an extent similar to that induced by zinterol in PTX treated cells (Figs.3B).Since a nearly identical dose-response curve to fenoterol was obtained after PTX treatment (Fig.3B),fenoterol-stimulated β2AR in mouse myocytes,as in rat myocytes,is uncoupled from Gi proteins.
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    Theoretically,it is possible that differences in agonist efficacy may masquerade an agonistspecific signaling selectivity,even if only one species of active receptor is present[8].In other words,an agonist with high intrinsic efficacy may activate all pathways;but a weak agonist,even at a maximal concentration,may activate only the most sensitive pathway,creating an apparent differential signaling[13].To test this possibility,we took advantage of a recently developed transgenic murine (TG4) model in which the human β2AR is overexpressed by~200-fold in a cardiac-specific manner[14],rendering a markedly enhanced receptor signaling strength.It is noteworthy that,in TG4 hearts,β1AR subtype is essentially non-functional in vivo[15],in isolated atria[6,14],or in both PTX-treated and untreated cardiac cells (data not shown).Although the cause for the loss of β1AR functionality is presently unknown,this phenotype affords an extra advantage for testing our hypotheses:the cellular response to any βAR agonist would be mediated almost exclusively through the nearly homogenous population of β2AR subtype.
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    As was the case in rat (Fig.2D)and in wild type littermates(Fig.3B),fenoterol(10-9 to 10-6M)induced a dose-dependent increase in cell contraction in TG4 myocytes,regardless of PTX treatment.More importantly,PTX treatment fully rescued the zinterol stimulated contractile response (Fig.3C)but had no potentiating effect on the contractile response to fenoterol(Fig.3D),indicating that an uncoupling of fenoterol stimulated β2AR from Gi protein still occurs in the receptor overexpression murine heart cells.Furthermore,zinterol(10-6M)markedly attenuated the fenoterol(10-6M)-induced increase in contraction amplitude from(312.4±40.8) to (156.9±14.8)% of control (n=4,P<0.01).The inhibition of the prevailing fenoterol effect by zinterol is consistent with the idea that both ligands act on the same population of β2AR.Taken together,our data indicate that β2AR agonist-directed differential G protein activation is preserved over several orders of magnitudes of receptor densities as well as of agonist concentrations in mammalian cardiac myocytes.This strongly supports the receptor “trafficking”hypothesis[8],but argues against the signaling strength hypothesis[8].
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    Agonist-directed“trafficking”of receptor stimulus implicates that a given receptor may possess distinct active conformation states when occupied by different agonists.To quantify how many active states of β2AR exist in intact cardiac myocytes,we devised a new strategy by examining the contractile response in the absence of PTX (a combination of Gs and Gi signaling) relative to the contractile response after PTX treatment (Gs signaling only).In principle,if a group of agonists,regardless of their efficacy,induced the same active state (in terms of G protein selectivity profile),plots for these agonists should follow the same trajectory.Conversely,if multiple active receptor species are induced,curves should no longer overlap.The number of separate clusters of curves would reflect the minimal number of active states of the receptor.As shown in Fig.4A,in rat myocytes,while the curves for zinterol,salbutamol and procaterol are clustered together,the plot for fenoterol is clearly divergent from the rest and is nearby the dashed line which represents a situation without Gi activation.A similar separation pattern was observed for zinterol and fenoterol in both WT and TG4 mice(Fig.4B).Hence,we consistently detected at least two active states of β2AR in all three physiological systems tested:fenoterol-stimulated β2AR favoring predominantly Gs interaction,and the other agonists stimulated β2AR activating both Gs and Gi signaling pathways (Fig.4C).This finding requires a reformulation of the current “two-state” receptor model[6] to accommodate a minimum of three receptor states including two active states.This multiple receptor state model is also supported buy recent observations on β2ARs[16] and α-adrenergic receptors[2] in transfected cell lines or in solublized systems.
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    In humans and animals,heart failure due to a variety of causes is characterized by a severely impaired βAR-mediated contractile response[17,18]associated with a selective down-regulation of β1AR(higher β21AR ratio)[17] and increases in Gi protein mRNA level,protein abundance and activity[19].Based on aforementioned findings,we hypothesized that a selective activation of the β2AR-Gs signaling pathway,bypassing Gi,by agents like fenoterol may open a new therapeutic avenue to rescue the βAR responsiveness in failing hearts.We tested this idea in two different heart failure models,namely,spontaneous hypertensive rats (SHR)[20] and salt-sensitive Dahl rats(DS)[21].Figs.5A& B show data obtained form SHR and age-matched Wistar-Kyoto (WKY)rats.While the positive inotropic effect of the β2AR agonist,zinterol,was markedly blunted in failing SHR hearts (Fig.5B),β2AR stimulation by fenoterol was still able to enhance contraction amplitude to an extend similar to that in age-matched controls (Fig.5A).Furthermore,PTX pretreatment completely restored the postitive inotropic effect of zinterol in SHR failing hearts (Fig.5B),indicating that the reduced response to β2AR stimulation by zinterol is mainly attributable to an upregulation of Gi signaling.Similarly,in DS model where the positive inotropic effect of zinterol was almost completely diminished (Fig.5D),contractile response to fenoterol remained intact relative to those in age-matched salt-resistant Dahl rat (DR) myocytes (Fig.5C).
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    In summary,we have demonstrated that ligand-directed receptor trafficking occurs in a physiologcal context,i.e.,native β2AR in rat and mouse hearts and human β2AR in genetically engineered murine hearts.The switching of the receptor's G protein selectivity by agonists has a tremendous range of therapeutic implications.For example,use of “signaling-specific”agonists and antagonists can enhance the desired effects while avoiding adverse side effects of therapeutic agents.Indeed,we demonstrated that fenoterol,the β2AR-Gs signaling-specific agonist,can rescue the deteriorated βAR contractile response (due to an enhanced Gi signaling)in failing rat hearts.In addition,these results may also shed new light on the biological significance of promiscuous coupling of receptor to G proteins.Not only the receptor entity or its phosphorylation status[22],but also the nature of its agonists determine the specificity of the receptor-G protein interaction and thereby of the transmembrane signal trans-duction.
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    Fig. Legends

    Fig. 1:Selective activation of cardiac β2-adrenergic receptor (AR) subtype by fenoterol,procaterol,salbutamol or zinterol.

    (A~F)Representative examples of continuous chart recordings of cell length in the presence of various βAR subtype agonists and antagonists.Cell shortening is indicated by an upward deflection.Panels A&B show that β1AR selective antagonist,CGP 20 712A(CGP,10-8M),blocked the β1-adrenergic contractile effect induced by norepinephrine (NE,10-7M) plus an α1-adrenergic antagonist,prazosin(10-6M),but not the equipotent effect induced by fenoterol (FEN,10-5M).Panels C~F show antagonistic effects of the β2AR blocker,ICI 115 881(ICI,10-7M),on contractile responses induced by fenoterol(FEN,10-5M),zinterol (ZINT,10-5M),salbutamol(SALB,5×10-5M)or procaterol(PROC,10-4M),respectively.(G)Average effects of CGP(10-8M).CGP completely reverses the positive inotropic effect of norepinephrine in the presence of prazosin,but cannot block those of fenoterol,procaterol,salbutamol,or zinterol.Dashed line marks the level of baseline contraction amplitude.Data reported are mean±SE.n=6~8 for each group.P<0.001 vs.without CGP.Data were obtained in adult rat ventricular myocytes.
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    Fig. 2:Differential potentiating effects of PTX treatment on the contractile responses to selective β2AR agonists.

    Dose-responses of contraction amplitude in rat ventricular myocytes are shown for salbutamol(A),procaterol(B),zinterol(C)and fenoterol (D) in the presence (solid symbols) and absence (open symbols) of PTX treatment.Note that PTX by itself had no significant effect on baseline contractionl ((5.29±0.13) and (5.57±0.13)% of rest cell length for PTX-treated group (n=126 cells) and non-treated group(n=126),respectively),but converted all the partial β2AR agonists used in A-C into full β2AR agonists.Note also that the potentiating effect of PTX occurred over a large agonist concentration range.Data were expressed as percent of control (%C).n=6~8 for each data point.Each cell was exposed to only one dose of agonist (to avoid possible receptor desensitization due to previous exposure to agonist).
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    Fig. 3:Receptor density and ligand-directed β2AR stimulus trafficking.

    Effects of zinterol and fenoterol on contraction amplitude in ventricular myocytes isolated from wild type (WT)(A and B) and transgenic mice overexprssing human β2AR (TG4)(C and D) were examined in the presence and absence of PTX treatment.Note that (i) zinterol elicited null inotropic response in both WT and TG4 cells unless Gi was prohibited by PTX;(ii)receptor overexpression alone failed to rescue a zinterol stimulated contractile response;(iii)fenoterol increased the contraction amplitude regardless of PTX treatment and receptor density;and (iv) the EC50 of zinterol (in PTX treated cells) and of fenoterol (in treated and untreated cells) was lower in TG4 versus WT cells,consistent with a higher receptor density in TG4 heart cells.Baseline contractility in TG4 and WT ventricular myocytes were (4.3±0.3)%(n=54) and (2.3±0.2)%(n=66,P<0.01) of rest cell length.The enhancement of basal contractility in TG4 cells is in agreement with previous observations (6,14) and supports the notion of spontaneous activation of β2AR in the absence of an agonist.
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    Fig. 4:Cardiac β2AR possesses at least three conformational states.

    Contractile responses in the absence of PTX (a combination of Gs and Gi signaling) are plotted as a function of those in the presence of PTX treatment (Gs signaling only). Dashed line indicates a situation without PTX-sensitive component;the vertical displacement form the dashed line reflects the level of Gi activation at a given level of Gs activation.The clustering and segregation of these curves will then reflect the similarity or distinction among different ligand-occupied receptor states.Panel A shows data of procaterol,salbutamol,zinterol and fenoterol in rat ventricular myocytes;Panel B shows data obtained from WT and TG4 mice.Panel C shows a model for ligand-directed,selective coupling of β2AR to Gs-or Gi-mediated pathways.Different agonists can selectively activate post-receptor events,presumably by stabilizing receptor in distinct active conformational states.In particular,fenoterol(Fen)-stimulated β2AR exclusively couples to Gs,whereas other agonists,such as zinterol(Zint),salbutamol(Salb)and procaterol (Proc),direct the β2AR to both signaling cascades mediated by Gs and Gi,respectively.The activation of β2AR-coupled Gi pathway provides a negative feedback to the Gs mediated contractile response(3,4).
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    Fig. 5:Fenoterol rescued the contractile response to β2AR stimulation in ventricular myocytes isolated from failing hearts of 18-24 month-old spontaneous hypertensive rats (SHR) and 4 month-old salt-sensitive Dahl rats (DS)[20,21].In

    both SHR (A) and DS (C) failing heart cells,fenoterol induced a robust increase

    in contraction amplitued to an extend similar to that in the age-matched controls,Wistar-Kyoto (WKY) and salt-resistant Dahl (DR) rat,respectively.Note
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    that the effect of zinterol in either SHR (B) or DS (D) was diminished relative to the response of nonfailing heart cells from corresponding control animals.Note

    also that the blunted responsiveness to zinterol was revived by PTX treatment.

    Data are presented as percent of control value (%C,mean±SE,n=5~8 cells from five

    hearts for each data point).Baseline contraction amplitued are (5.42±0.23)
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    (n=38 cells) and (4.66±0.25)% of rest cell length (n=36 cells) for SHR and WKY,respectively,and (4.8±0.36) and (4.05±0.5)% of rest cell length for DS

    (n=32 cells) and DR (n=30 cells),respectively.PTX pretreatment had no

    significant effect on baseline contraction amplitude (4.88±0.14)(n=17) and

    (5.52±0.34)% of rest cell length (n=18) for SHR and WKY,respectively).■
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    (1994);R-P.Xiao,H.A.Spurgeon,F.O'Connor,E.G.Lakatta,J.Clin Invest.94,2051(1994)].To

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    al.Am.J.Physiol.41,H590(1997)].In some experiments,aliquots of cells were incubated
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    with PTX(1.5 μg/ml,at 37℃ for at least 3 h) to abrogate Gi protein function via

    ribosylation,as previously described[3,4].After PTX treatment,cells were kept at

    the room temperature for the rest of the experimental day (6~8 h).PTX-treated cells

    were compared with myocytes from the same heart which had been kept at 37℃ in the

    absence of PTX for an equal time.Cells were then perfused with Hepes buffer solution
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    consisting of (in mM):1.0 CaCl2,137 NaCl,15 dextrose,1.3 MgSO4,1.2 NaH2PO4,20

    Hepes,pH 7.4,and were electrically stimulated at 0.5 Hz at 23℃.Cell length was

    monitored from the brightfield image of the cell by an optical edge-tracking method

    using a photodiode array (Reticon Model 1024 SAQ) with a 3 ms time resolution.Cell
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    contraction was indexed by the percent reduction of cell length following electrical

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    classical adenylyl cyclase-cAMP-protein kinase A (PKA) cascade,because the

    β2AR-stimulated increase in L-type Ca2+ channel current or contractile response is

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    a peptide PKA inhibitor,PKI

    [V.A.Skeberdis,J.Jurevicius,R.Fischmeister,J.Pharmacol.Exp.Ther.283,452(1997)].The
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    β2AR-stimulated Gi signaling is thought to be an negative feedback mechanism to

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    A.Grove,Br.J.Clin.Pharmacol.43,9,(1997).

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    activation of Gs and Gi signaling by zinterol and fenoterol.Firstly,fenoterol,an

    extremely weak agonist for β2AR-Gi signaling,was as efficacious (if not more

    efficacious) as the other agonists in activating the β2AR-Gs

    signaling.Secondly,zinterol-stimulated Gi signaling occurred even at low level of
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    signaling strength,when only a small fraction of β2ARs were occupied (e.g.,~20%

    at 10-8 M,assuming a Kd of 40 nM for zinterol binding to β2AR)

    [K.P.Minneman,A.Hedberg,P.B.Molinoff,J.Pharmacol.Exp.Ther.211,502(1979)].These results suggest that fenoterol- and zinterol-occupied β2AR differ qualitatively with respect to their propensity of activating Gs and Gi signaling pathways.
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    [20]For a relevant animal model of clinical hypertensive heart disease and

    heart failure,we studied the spontaneously hypertensive rat (SHR)and age-matched
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    Wistar-Kyoto (WKY) controls at an age (18-24months)when SHR with cardiac

    hypertrophy showed physical signs of heart failure (e.g.resting

    tachycardia,tachypnea,pleural and/or pericardial effusions)

    [J.M.Pfeffer,M.A.Pfeffer,M.C.Fishbein,E.D.Froloch,Am.J.Physiol.237,H461

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    (1991)].Blood pressures were measured weekly in every animal by the tail-cuff

    method in conscious non-sedated animals pre-warmed to 37℃.At 3 months-of-age,SHR were clearly hppertensive,and at the time of study SHR had systolic blood

    pressures of(182±21)mmHg(vs WKY:(121±11)mmHg,P<0.05,n=21).Cardiac hypertroph

    was confirmed by significant increases:1)in heart weight (wet)/body weight
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    (mg/g;SHR:5.10±0.08 vs WKY:3.8±0.5,P<0.05,n=13),2)in echocardiographic

    measurements of left ventricular wall thickness (e.g.posterior wall

    thickness,mm;SHR:2.1±0.3 vs WKY:1.6±0.3,P<0.05,n=10),3)in electrically

    determined measurements of cell capacitance (pF;SHR:270±43 vs WKY 180±30,P<0.05,n=44),4)cross-sectional area of cells computed from images of

    transmitted light microscopy (μm2,SHR:296±78 vs WKY:135±58,P<0.001,n=256).
, 百拇医药
    [21]Six week old Dahl salt-sensitive (DS)(SS/JrHsD) and resistant

    (DR) SR/JrHsd) rats were obtained from Harlan Sprague Dawley

    Inc,Indianopolis,IN.Animals were given normal(0.2%) or high (8%) NaCl diets

    (ICN Pharmaceuticals,Costa Mesa,CA).Starting from week 4 of high NaCl diet,animals were given 8% NaCl diet 5 days a week and 0.2% NaCl diet 2 davs a week

    (days 5 and 7).At baseline,systolic blood pressure (SBP) in 12 DS and 12 DR
, 百拇医药
    was 106±2 and 108±2 mmHg,respectively.In DS on 8% NaCl diet SBP gradually rose

    and reached plateau on week 2 (161±7 mmHg,n=6,P<0.001 vs.baseline).In

    DR after 2 weeks of 8% NaCl diet SBP was 118±4 mmHg (n=6,P<0.001 vs.DS at

    week 2).SBP in DS after 2 weeks of 02% NaCl diet was 114±3 mmHg (n=6,P<0.001

    vs.DS on 8% NaCl diet).On week 8 of high NaCl diet,DS exhibited symptoms of congestive heart failure:rapid and labored respiration,loss of physical
, http://www.100md.com
    activity and decrease of body weight.When sacrificed,pathological examination revealed pleural effusion and ascites.The heart/body weight ratios in DS on 8%

    and 0.2% NaCl diets were 6.9±0.1 and 3.7±0.1(P<0.01).The lung/body weight

    ratios were 11.4±0.8 and 4.1±0.3(P<0.01),respectively.In DR on 8% NaCl diet

    heart/body and lung/body wight ratios were 4.0±0.1 and 3.9±0.1 (P<0.01 vs.DS
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    on 8% diet for both parameters).

    [22]Y.Daaka,L.M.Luttrell,R.J.Lefkowitz,Nature 390,88(1997).

    [23]The authors would like to thank Dr.Michael Stern for his critical reading of

    the manuscript;Drs.Robert Lefkowitz and Walter J.Koch for kindly supplying us TG4

    mice;and Dr.Harold Spurgeon,Bruce Ziman and Lucy Jiang for excellent technical

    support.This work was suppoted by NIH intramural research programs (RPX,EGL and

    HC) and extramural grants (RAB and CWB)., http://www.100md.com