Alpha-Adrenergic Receptors in Rat Myocardium

Transcription

1 Alpha-Adrenergi Reeptors in Rat Myoardium Identifiation by Binding of [ 3 H]Dihydroergoryptine 721 R. SANDERS WILLIAMS AND ROBERT J. LEFKOWITZ SUMMARY [ 3 H]Dihydroergoryptine ([ 3 H]DHE) binds to sites in membranes derived from rat myoardium that have the harateristis expeted of a-adrenergi reeptors. The binding is saturable with 41 fmol [ 3 H]DHE bound per mg of protein and of high affinity with KD = 2.9 niu. The binding is rapid and readily reversible. Adrenergi agonists ompete with [ 3 H]DHE for binding in the order: epinephrine > norepinephrine» isoproterenol; and adrenergi antagonists ompete for binding in the order: phentolamine» propranolol. For omparison, ( )[ 3 H]dihydroalprenolol [( )[ 3 H]DHA] was used to bind to sites in the same membrane preparations having harateristis of /{-reeptors. The number and affinity of /?-reeptors were quite similar to those of the a-reeptors with 46 fmol ( )[ 3 H]DHA per mg protein bound at saturation and KD = 2.5 nm. These tehniques allowed identifiation of both ft- and o-adrenergi reeptors in membranes derived from isolated atria, right ventriular free walls, and left ventriles inluding interventriular septa. This is the first report doumenting diret identifiation of myoardial a-reeptors by radioligand-binding tehniques and omplements the literature previously reporting myoardial inotopi and eletrophysiologial responses to a-adrenergi stimulation. AHLQUIST'S' original subdivision of responses to ateholamines into two distint ategories, a and /?, has proven to be an extremely useful onept in the understanding of adrenergi hormones and drugs. Early work in this area seemed to indiate that the myoardial response to adrenergi stimulation was mediated exlusively through js-reeptors. However, with the development of adrenergi agonists and antagonists having greater speifiity for either the a- or the /8-reeptor, a large body of data has aumulated whih suggests that the heart demonstrates responses mediated through a-reeptors as well as /S-reeptors. Several investigators have demonstrated that a- adrenergi agonists suh as phenylephrine or the ombination of epinephrine plus propranolol stimulate myoardial responses suh as inreased ontratility, inreased funtional refratory period, inreased duration of ation potential, and dereased rate of phase 4 depolarization. 2 " 22 In addition, these responses are bloked by speifi a-adrenergi antagonists, suh as phentolamine, but not by /?-adrenergi antagonists. Radioligand binding studies have been used in the past few years to identify adrenergi reeptors in numerous tissues, inluding the identifiation of From the Departments of Mediine and Biohemistry, Duke University Medial Center, Durham, North Carolina. Supported by Health, Eduation, and Welfare Grants HL16037 and HL20339, and by a Grant-in-Aid from the North Carolina Heart Assoiation. Dr. Lefkowitz is an Investigator of the Howard Hughes Medial Institute. Address for reprints: Dr. Robert J. Lefkowitz, Departments of Mediine and Biohemistry, Duke University Medial Center, Durham, North Carolina Reeived November 10, 1977; aepted for publiation June 20, /^-reeptors in rat and dog myoardium 2324 The identifiation of binding sites in the myoardium having harateristis expeted of a-reeptors would provide further evidene for the existene of myoardial a-reeptors, and this tehnique ould be used to study adrenergi modulation of ardia funtion at the moleular level. The radioligand [ 3 H]dihydroergoryptine ([ 3 H] DHE, a potent a-adrenergi antagonist) has been used to identify binding sites that have the harateristis expeted of a-adrenergi reeptors 25 ' 26 in membrane frations derived from smooth musleontaining tissues suh as rabbit uterus 27 and anine aorta, 28 as well as rat brain 29 and human platelets. 30 Aordingly, we undertook studies to determine if suh radioligand-binding tehniques ould be used to doument the existene of a-adrenergi reeptors in the mammalian myoardium. In this ommuniation we report the appliation of these methods to demonstrate and haraterize a-adrenergi reeptors in partiulate frations derived from rat myoardium. Methods Radioligands [ 3 H]DHE (speifi ativity, 25 Ci/mmol) was obtained from New England Nulear and was shown to be homogeneous and indistinguishable from unlabeled dihydroergoryptine by thin layer hromatography in two different solvent systems. 27 Stored at 20 C in ethanol and proteted from light, it is stable for up to 6 months. Immediately prior to use, appropriate amounts of stok solution were diluted with deionized water ontaining 2.5 mm HC1 and

2 722 CIRCULATION RESEARCH VOL. 43, No. 5, NOVEMBER % ethanol. This mixture was further diluted 6-fold by addition to the final inubation volume suh that ethanol onentration in the final assay was 1%. Ethanol in final onentrations up to 4% has been shown to have no effet on speifi binding of [ 3 H] DHE. 27 (-)[ 3 H]DHA (speifi ativity, 33 Ci/mmole) was also obtained from New England Nulear and found to be homogeneous and idential to the parent ompound by thin layer hromatography. 31 Immediately prior to use, appropriate dilutions of stok solution stored in ethanol were made with deionized water. Membrane Preparation Male CD strain rats from Charles River weighing g were killed by a blow to the head and the hearts rapidly exised and plaed in buffer (4 C) onsisting of surose, 0.25 M; Tris-HCl, 5 HIM; MgCl2, 1 mm; ph 7.4. Fat and great vessels were trimmed and removed. The hearts were then finely mined and homogenized in old buffer with a Teflon pestle (Thomas) in a Potter-Elvejhem homogenizer driven at high speed. Pooled hearts from four to eight rats were used for eah experiment. Exept where speified, both atria and both ventriles were used for the preparation of membranes. For the experiments in whih reeptors were studied in different anatomi regions of the heart, the hearts were plaed in a shallow plasti dish filled with old buffer and the atria separated from the ventriles by utting along the atrioventriular sulus. The interventriular septum and left ventriular free wall were then separated from the right ventriular free wall by grasping the septum with foreps and removing the right ventriular free wall as seen from above. The hambers were then kept separate and membranes prepared as stated. The homogenates were filtered through a single layer of heeseloth, then entrifuged at 1000 g for 10 minutes in a Sorvall RC-2B entrifuge. The pellets onsisting of fibrous tissue and high density debris were disarded and the supernatant extrats then reentrifuged at 40,000 g for 10 minutes. The resulting pellets were washed twie in old inubation buffer onsisting of 50 mm Tris-HCl, 10 mm MgCl, ph 7.5, by resuspension and entrifugation, and then finally resuspended in inubation buffer at a onentration of 1-2 mg of protein per ml. Protein was determined by the method of Lowry et al. 32 Binding Assay Exept where otherwise stated, membranes were inubated for 18 minutes at 25 C with [ 3 H]DHE or (-)[ 3 H]DHA in a total volume of 900 ja of inubation buffer. Inubations were ended by rapid vauum filtration of the entire mixture through Whatman GFC glass fiber filters. The filters were immediately washed with 20 ml of inubation buffer (25 C) for [ 3 H]DHE binding or 10 ml of inubation buffer (4 C) for (-)[ 3 H]DHA. In separate experiments, these different wash volumes were shown to optimize the "speifi" binding for eah respetive radioligand. Filters were air dried overnight, plaed in a Triton X-100/toluene-based fluor, and radioativity was determined in a Pakard liquid sintillation spetrometer at an effiieny of 50%. "Speifi" binding of [ 3 H]DHE was defined as that radioativity bound to membranes whih ould be displaed by 10 ym phentolamine. "Speifi" binding of [ 3 H]DHE ranged from 35% to 60% of total ounts bound per filter. However if ounts nonspeifially adsorbing to the filters (as alulated by pouring membranefree inubation mixtures over filters and subjeting these to 20-ml washes idential to that used for membrane-ontaining inubation mixtures) are subtrated, the "speifi" binding ranged from 50% to 75% of the total ounts bound to the membranes themselves. For (-)[ 3 H]DHA, "speifi" binding was defined as that displaed by 10 JUM (±)propranolol and ranged from 50% to 68%. Counts bound to the filters in the absene of membranes (filter blank) ranged from 0.4% to 0.9% of the total ounts filtered after the filters were washed in a manner idential to that used in the presene of membranes. In a typial experiment, total binding to membranes was 3.0% of total ounts filtered, binding to membranes in the presene of 10" 5 M phentolamine was 1.4% of total ounts filtered, and speifi binding was therefore 1.6% of total ounts filtered or 53% of total ounts bound. We all this "53% speifi binding." If, however, a "filter blank" of 0.6% of total ounts filtered is subtrated from total binding and from binding in the presene of 10~ 5 M phentolamine, thus estimating the binding to the membranes themselves, then the "speifi binding to the membranes" ould be alulated as ( )/2.4 = 67%. The presene or absene of phentolamine had no effet on the "filter blank." In eah experiment, binding was determined in tripliate idential inubation mixtures for eah data point. Results Number and Affinity of Binding Sites; Kinetis of Binding The binding of [ 3 H]DHE to ardia membrane frations was found to be saturable and of high affinity (Fig. 1). Sathard analysis 33 of these data revealed a linear plot suggesting a single lass of binding sites with 41 fmol of [ 3 H]DHE bound per mg protein at saturation and a KD of 2.9 nm alulated from the negative reiproal of the slope of the line (Fig. 1, inset). Kineti analysis of the binding of [ 3 H]DHE to ardia membranes showed it to be rapid with halfmaximal binding by 2.5 minutes at 25 C. Binding remained at steady state for as long as 34 minutes (Fig. 2A). The binding of [ 3 H]DHE was readily

3 pun [ 3 H]DHE AND a-receptors IN RAT HEART/ Williams and Lefkowitz DD T3 C s >, o ^ ^ 0) B 30 Q. Ql / H H <V ergor ypt 0 jroli 1-1 a \ "0 E 01 o T3.C Q 2 i E 10 " / i ^l0 _/ i 8 t A «l2 - \. \ TIME (minutes) 0 fa 0 10? _ phj D'hydroergoryprine Bound (f moi/mq prot«n) I I i i r O 22 [f 3 HJ Dihydroergoryptme (nm) FIGURE 1 Speifi binding of fhjdhe to rat myoardial membranes as a funtion of inreasing onentrations of l^hjdhe. Speifi binding is defined as fh] DHE binding displaeable by 10~ b M phentolamine. Eah value is the mean of tripliate determinations in three separate experiments. Beause of the diffiulty involved in repliating the exat onentrations of f^hjdhe at values greater than 9 nm, losely related onentrations were grouped and the horizontal bars represent the range of onentrations that were inluded in the alulation of eah value. The vertial bars represent the standard error of the mean for eah value. Inset: Sathard plot of f^hj'dhe binding to rat myoardial membranes. The ratio (B/F) of bound fhjdhe (fmol/mg protein) to free f^hjdhe (nm) is plotted as a funtion of the fhjdhe bound per mg protein. The number of binding sites is alulated from the interept with the x axis. The K D was alulated from the negative reiproal of the slope as determined by linear regression analysis. reversed by the addition of 1CT 4 M phentolamine (Fig. 2B). We observed somewhat greater binding at equilibrium in the kineti experiments as ompared to the saturation experiments. In 10 saturation or ompetition experiments involving DHE at onentrations near 1.3 nm, the mean binding ± SEM was 14.6 ± 2.6 fmol/mg protein. In omparison, in five experiments examining kinetis at 1.3 nm DHE, the mean binding ± SEM was 20.0 ± 6.9 fmol/mg protein. This differene was not statistially signifiant. In the experiments analyzing reversal of [ 3 H] DHE binding by the addition of 10~ 4 M phentolamine, we observed no derement in binding at 30 and 60 seonds (Fig. 2B). We do not believe this represents the physiologial reality, but suggest, rather, that the preision of the methods was not suffiient to detet small redutions in binding at these early points. A seond possibility would be that inadequate mixing of the added phentolamine ourred (100 y\ added to 7.1 ml inubation volume) despite gentle vortexing of the tubes TIME (minutes) FIGURE 2 Kinetis of f'hjdhe binding to ardia membranes. A: f^hjdhe binding to ardia membranes as a funtion of time. Cardia membranes were inubated in large volumes (7.1 ml), and at the speified intervals after the addition of fhjdhe (1.3 nm), 1.0-ml samples were removed and filtered as desribed in Methods. Speifi binding was defined as the differene between inubation mixtures with lo'^mphentolamine and otherwise idential mixtures without phentolamine. The results expressed here are the means of tripliate determinations from three separate experiments. Eah time point was determined in at least two of the three experiments. B: Reversibility of f^hjdhe binding to ardia membranes. Membranes were inubated with fhjdhe (1.3 nm) in 7.1-ml inubation volumes for 18 minutes at 25 C, at whih time exess phentolamine was added to all tubes to yield a final onentration of 10'" M. The time of phentolamine addition is defined here as t = 0 minutes. Speifi binding was then determined as the differene in f^hjdhe bound between tubes originally ontaining 10~ 5 M phentolamine and tubes without phentolamine and plotted as a funtion of time. The values expressed here are the means of tripliate determinations from two separate experiments, exept for the points at 1/2, 1, and 28 minutes whih were performed in tripliate in one experiment. Speifiity of Binding Adrenergi agonists ompeted with [ 3 H]DHE for the binding sites in the order expeted of an a- reeptor, i.e., epinephrine > norepinephrine > isoproterenol (Fig. 3). Stereospeifiity, another harateristi expeted of adrenergi reeptors, was demonstrated by the onsiderably greater poteny

4 724 CIRCULATION RESEARCH VOL. 43, No. 5, NOVEMBER 1978 O (-)Epmepnnne 00 A (-) Norepinephrine o 100 D (-)Isoprorerenol ( + ) Epinephrine * ( + ) Norepinephrine Q "if s Log O [Adrenergi Agonist] M FIGURE 3 Competition by adrenergi agonists for binding of phjdhe. Cardia membranes were inubated with fhjdhe (1.3 nm) in the presene or absene of the indiated agonists and speifi binding was determined. "100% Inhibition" refers to the inhibition of fhjdhe binding indued by 10~ b M phentolamine. Eah value is the mean of tripliate determinations from the number of experiments indiated beside eah point. of the ( )stereoisomers of epinephrine and norepinephrine as ompared with the (+)stereoisomers in ompeting for the binding of [ 3 H]DHE (Fig. 3). Adrenergi antagonists also ompeted with the expeted speifiity, phentolamine exhibiting markedly greater affinity for the binding sites than propranolol (Fig. 4). The onentrations of these and other ompounds neessary to ompete for 50% of the speifi binding of [ 3 H]DHE are expressed in Table 1. Comparison of a- and /8-Reeptors For omparison, binding of ( )[ 3 H]DHA to ardia membranes was measured using the idential membrane preparation and inubation onditions 00 IOO I b o Phentolamine (±)Propranolol Log O [Adrenergi Antagonist] M FIGURE 4 Competition by adrenergi antagonists for binding of fhjdhe. For explanation of terms, see legend to Figure TABLE 1 Inhibition offhjdhe Binding to Cardia Membranes by Adrenergi Agents Agonists (-)Epinephrine (-) Norepinephrine (+) Epinephrine (-) Pheny lephrine (+) Norepinephrine (-)Isoproterenol Antagonists and related ompounds Phentolamine (±)Propranolol Serotonin Dopamine ECM (/*M) Poteny relative to ( )epinephrine used to haraterize the binding of [ 3 H]DHE. ( )[ 3 H]DHA has previously been shown to label sites indistinguishable from physiologial /?-reeptors in both rat and dog ardia membranes, 23 ' 24 though under experimental onditions somewhat different from those employed in the urrent studies. The number of binding sites for ( )[ 3 H]DHA was 46 fmol bound per mg of membrane protein, and a KD of 2.5 nm was alulated from the negative reiproal of the slope of the Sathard plot (Fig. 5). These values were quite similar to those deter- (-)f*hj Ohydroolprenolol Bound (fmol/mg protein) [(-)[ 3 H] Dihydroalprenolol] (nm) FIGURE 5 Speifi binding of ( l^hjdha to ardia membranes as a funtion of inreasing ( )^H]DHA. Speifi binding is defined as ( )[*H]DHA binding displaeable by 10' 5 M (±) propranolol. Eah value is the mean of tripliate determinations from the number of experiments stated below eah point. Just as in Figure 1, the horizontal bars represent the range of onentrations of ( Jl^HJDHA used for eah value, and the vertial bars represent the standard error of the mean. Inset: Sathard plot of ( JfHJDHA binding to rat myoardial membranes with the ratio of bound ( JfHJDHA (fmol/mg protein) to free (-ffhjdha (nm) plotted as a funtion of bound (-ifhjdha (fmol/mg protein). The K D is alulated by the method desribed in Figure 1.

5 [ 3 H]DHE AND a-receptors IN RAT HEART/ Williams and Lefkowitz 725 mined for the sites binding [ 3 H]DHE in our membrane preparation. The number and affinity of /J-reeptors in rat ardia membranes as determined in this study differ somewhat from the values previously reported from this laboratory. 23 While we have no definitive explanation for these differenes, distintly different experimental onditions (larger inubation volume, lower reeptor onentration in the final membrane suspension, lower temperature) were employed and probably aount for the differene in results. It should be noted that high onentrations of (-)[ 3 H]DHA (greater than 15 nia) appeared to label a seond order of binding sites with a muh lower affinity. However, the auray of data obtained at these very high onentrations was poor (very low perentage of "speifi binding"), and this low affinity site did not demonstrate the harateristis, suh as stereospeifiity, expeted of /8-adrenergi reeptors. Experiments not shown here did onfirm that (-)[ 3 H]DHA binding to the high affinity site demonstrated harateristis expeted of /?-reeptors, i.e., stereospeifiity. For these reasons, only points on the saturation urve obtained at (-)[ 3 H] DHA onentrations less than 15 nm were used in the Sathard analysis quantitating number of binding sites and K D. We do not have a lear explanation as to why the urrent experimental methods seemed to detet this low affinity, propranolol-displaeable, but "nonspeifi" site when this phenomenon was not observed in earlier experiments. 23 However, this again presumably relates to differenes in experimental tehniques ited above. Anatomi Loalization of a- and /}-Adrenergi Reeptor Binding Sites Using [ 3 H]DHE and (-)[ 3 H]DHA, we sought to determine the relative densities of the two lasses of binding sites in several regions of the heart inluding the atria, the right ventriular free wall, and the left ventriular wall. In these studies the interventriular septum was inluded as part of the left ventrile. The results indiate that both lasses of binding TABLE 2 Binding of fhjdhe and (-)fhjdha sites are demonstrable in eah region of the heart. In three experiments eah involving pooled hearts from 8-20 rats, no signifiant differene was observed between the left and right ventriles in the binding of [ 3 H]DHE (Table 2). In two further experiments measuring the binding of ( )[ 3 H]DHA, membranes of left ventriular origin demonstrated a somewhat greater number of binding sites than those derived from right ventriles (Table 2). No differenes in K D were observed for either lass of binding sites. While speifi binding to atrial membranes was observed for both (-)[ 3 H]DHA and [ 3 H]DHE, the small quantity of membrane protein available despite the use of up to 20 rats did not allow aurate quantifiation of KD or total number of sites (data not shown). Disussion Validation of the Existene of Myoardial a-reeptors [ 3 H]DHE was used in these experiments to identify binding sites in a partiulate membrane suspension derived from rat myoardium that have the harateristis expeted of a-adrenergi reeptors. The binding is of high affinity, saturable, stereospeifi, and demonstrates forward and reverse kinetis appropriate for the binding of radioligand to adrenergi reeptors. In addition, the a-adrenergi agonists epinephrine and norepinephrine were 40 to 400-fold more potent in ompeting for the binding of [ 3 H]DHE than the /S-speifi agonist, isoproterenol, and the a-adrenergi antagonist phentolamine was 2500-fold more potent than the /^-speifi antagonist, propranolol. These harateristis learly distinguish these binding sites from those sites identified in myoardial membrane frations by ( )[ 3 H]DHA whih appear to represent /S-adrenergi reeptors. 23 ' 24 While we do not report new physiologial or biohemial responses orrelating with the harateristis of radioligand binding, numerous prior reports doument physiologial myoardial responses to drug ombinations that speifially stimulate a- adrenergi reeptors. 2 " 22 To equate these observa- to Anatomi Regions of the Heart Anatomi region (nm) Sites (fmol/mg protein) [ 3 H]DHE Right ventrile Left ventrile (-)[ 3 H]DHA Right ventrile Left ventrile NS = not signifiant. * Unpaired -test. P < 0.05*

6 726 CIRCULATION RESEARCH VOL. 43, No. 5, NOVEMBER 1978 tions in broken ell preparations with the ontrol mehanisms of physiologial responses in vivo would be somewhat premature. However, the striking similarity between the harateristis of DHE interation with ardia membrane binding sites and the pharmaologial harateristis of a-adrenergi reeptors strongly supports the ontention that DHE identifies a-reeptors at the moleular level. An inrease in ontratile fore of the myoardium is the most onsistently reported response to a-speifi stimulation, 2 " 17 although some investigators have reported a negative inotropi response. 34 ' 35 A different mehanism of ation from the positive inotropi effets of /?-adrenergi stimulation is suggested by several findings: (1) the time ourse of the development of the positive inotropi effet differs between a- and /J-stimulation 13 ' 14 ' 36 (2) the eletrophysiologial sequelae of a-stimulation are an inreased duration of the ation potential, 20 inreased funtional refratory period, 3 ' 6> l9 and dereased automatiity, 20 ' 21> 22 all of whih are in opposite diretion to the effets of /J-stimulation; (3) most investigators report either no hange or a redution in yli AMP in response to a-speifi stimulation. 13 " ' 36 ' 37 Previous Attempts to Identify Myoardial a-reeptors by Radioligand Binding Previous studies using [ 3 H]phenoxybenazmine 38 to label binding sites in myoardial preparations have been reported. The haloalkylamine phenoxybenzamine is widely known to be a nonspeifi alkylating agent that binds ovalently to a variety of membrane omponents other than the a-reeptor, 39 and the tritium-labeled ompound is of suh low speifi ativity (33 mci/mmol) as ompared to [ 3 H]DHE (25 Ci/mmol) that it is extremely unlikely that binding sites for this radioligand represent the physiologial a-reeptor. No kineti studies, saturation urves, or ompetition urves with adrenergi agents were reported. A preliminary report of binding of [ 3 H]DHE to ardia membranes has been published. 40 However, the data presented in this paper are diffiult to evaluate in terms of speifi labeling of a-reeptors, sine no experiments doumenting appropriate kinetis, stereospeifiity, or order of agonist ompetition were reported. Physiologial Signifiane of Myoardial a-reeptors Baroreeptor reflexes ourring in vivo make lear separation of the primary myoardial responses to a-and /?-adrenergi stimulation and blokade more diffiult than in isolated heart preparations. In intat animals and in man, the myoardial /8-adrenergi responses seem learly dominant over any diret responses mediated by a-reeptors when mixed agonists are employed. This phenomenon for years obsured the very existene of myoardial a-adrenergi responses. However, the distint possibility exists that the a-mediated myoardial responses desribed earlier beome more important in ertain irumstanes, most notably in the presene of /?-adrenergi blokade. In addition, reports that other fators, suh as hypothyroidism, 14 ' 38 dereased rate of paing, 4 ' 6> 7 ' 10 ' 41 and inreased alium onentration, 35 may potentiate the response to a-speifi stimulation suggest that myoardial a-reeptors may assume partiular signifiane in ertain disease states as well. Potential Future Usefulness of Diret Radioligand Binding Tehniques to Measure a-adrenergi Reeptors Altered adrenergi ontrol of myoardial funtion may be impliated in a variety of pathologial onditions inluding ongestive heart failure, hypertension, hypo- and hyperthyroidism, and sudden arrhythmi death. Radioligand binding tehniques omplement lassial pharmaologial studies by providing an assessment of the harateristis of reeptor binding at the moleular level. Espeially by providing for the simultaneous assessment of both /?- and a-reeptors, these methods may prove useful in assessing hanges in reeptor harateristis assoiated with various disease states. On a more basi level, the possibility has been suggested that myoardial a- and /J-reeptors are interonvertible forms of the same maromoleule. 38 ' 42 The apaity to assess diretly both speies of reeptors after interventions known to influene the relative magnitudes of the respetive physiologial responses to a- and ^-stimulation should be of use in resolving this provoative issue. Referenes 1. Ahlquist RP: A study of the adrenotropi reeptors. Am J Physiol 135: , Govier WC: Myoardial alpha-adrenergi reeptors and their role in the prodution of a positive inotropi effet by sympathomimeti agents. J Pharmaol Exp Ther 159: 82-90, Govier WC: A positive inotropi effet of phenylephrine mediated through alpha-adrenergi reeptors. Life Si 6: , Mugelli A, Ledda F, Mantelli L: Frequeny dependene of the alpha-adrenoeptor-mediated positive inotropi effet in guinea pig heart. Eur J Pharmaol 36: , Verma SC, MNeill JH: Biohemial and mehanial effets of phenylephrine on the heart. Eur J Pharmaol 36: , Benfey BG: Charaterization of alpha-adrenoeptors in the myoardium. Br J Pharmaol 48: , Endoh M, Shumann HJ: Frequeny dependene of the positive inotropi effet of methoxamine and naphazoline mediated by alpha-adrenoeptors in the isolated rabbit papillary musle. Naunyn Shmiedebergs Arh Pharmaol 287: , Endoh M, Shumann HJ, Krappitz N, Hillen B: Alphaadrenoeptors mediating positive inotropi effets on the ventriular myoardium: Some aspets of struture ativity

7 [ 3 H]DHE AND a-receptors IN RAT HE ART/ Williams and Lefkowitz 111 relationship of sympathomimeti amines. Jap J Pharmaol 26: , Shumann HJ, Endoh M, Wagner J: Positive inotropi effets of phenylephrine in the isolated rabbit papillary musle mediated both by alpha and beta-adrenoeptors. Naunyn Shmiedebergs Arh Pharmaol 284: , Shumann HJ, Endoh M: Alpha-adrenoeptors in the ventriular myoardium: Clonidine, naphazoline, and methoxamine as partial alpha-agonists exerting a ompetitive dualism in ation to phenylephrine. Eur J Pharmaol 36: , Fujimoto L, Borda L, Shuhleib R, Corr P, Henry P: Alphaadrenergi mediated enhanement of ventriular inotropy (abstr). Cirulation 53/54: , Wagner J, Endoh M, Reinhardt D: Stimulation by phenylephrine of adrenergi alpha- and beta-reeptors in the isolated perfused rabbit heart. Naunyn Shmiedebergs Arh Pharmaol 282: , Osnes JB: Positive inotropi effet without yli AMP elevation after alpha-adrenergi stimulation of perfused hearts from hypothyroid rats. 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