JOURNAL OF EXPERIMENTAL ZOOLOGY 293:99–105 (2002) Identification of a1-Adrenergic Receptorsand Their Involvement in PhosphoinositideHydrolysis in the Frog Heart ANTIGONE LAZOU,1* CATHERINE GAITANAKI,2 SPIROS VAXEVANELLIS,1 AND ANASTASIA PEHTELIDOU11Laboratory of Animal Physiology, Department of Zoology, School of Biology,Aristotle University of Thessaloniki, Thessaloniki 54006, Greece 2Department of Animal and Human Physiology, School of Biology,University of Athens, Athens 15784, Greece The aim of this study was to characterize a1-adrenergic receptors in frog heart and to examine their related signal transduction pathway. a1-Adrenergic binding sites were studied inpurified heart membranes using the specific a1-adrenergic antagonist [3H]prazosin. Analysis of thebinding data indicated one class of binding sites displaying a Kd of 4.19 7 0.56 nM and a Bmax of14.66 7 1.61 fmol/mg original wet weight. Adrenaline, noradrenaline, or phenylephrine, in thepresence of propranolol, competed with [3H]prazosin binding with a similar potency and a Ki value ofabout 10 mM. The kinetics of adrenaline binding was closely related to its biological effect.
Adrenaline concentration dependently increased the production of inositol phosphates in the heartin the presence or absence of propranolol. Maximal stimulation was about 8.5-fold, and the half-maximum effective concentration was 30 and 21 mM in the absence and presence of propranolol,respectively. These data clearly show that a1-adrenergic receptors are coupled to the phosphoinosi-tide hydrolysis in frog heart. To our knowledge, this is the first direct evidence supporting thepresence of functional a1-adrenergic receptors in the frog heart. J. Exp. Zool. 293:99–105, 2002.
r 2002 Wiley-Liss, Inc.
Noradrenaline and adrenaline play important metabolic responses (Terzic et al., '93; Li et al., '97; Varma and Deng, 2000). a1-Adrenergic stimu- throughout the body. The adrenoceptors through lation may also have longtime effects on cardiac which these compounds act are subdivided into structure and function, since exposure to a1- three families (a1, a2, b) based on their pharma- adrenergic agonists leads to activation of growth- cology, structure, and signaling mechanisms (Hie- related gene expression (Knowlton et al., '93; ble et al., '95). Within each family subtypes exist Sugden and Clerk, '98). The effects of a-adrenergic that have been defined structurally by molecular stimulation on the amphibian heart are far less cloning of their respective genes and pharmacolo- understood and are still a matter of controversy.
gically by the interactions of subtype-selective Whereas some authors have failed to find an a- agonists and antagonists with these receptors.
adrenoceptor-mediated inotropic response (Morad Most of these studies have been carried out using et al., '78; Soustre and Rakotonirina, '81), others mammalian adrenoceptors, and there is relatively suggest that a-adrenergic receptors may partici- little information available on adrenoceptors in pate in the positive inotropic action of sympatho- amphibians. The presence of these subtypes of adrenoceptors and their role in lower vertebrate Niedergerke and Page, '77; Petroff et al., '94). It heart activity have been the subject of debate in has also been questioned whether cardiac a- the past, particularly with reference to theirfunctional and morphological relationships (Ask, *Correspondence to: Dr. Antigone Lazou, Laboratory of Animal '83; Chiu and Sham, '85; Sham et al., '87).
Physiology, Department of Zoology, School of Biology, Aristotle In mammalian cardiac cells, responses to a University of Thessaloniki, Thessaloniki 54006, Greece. E-mail: adrenergic stimuli include rapid changes in con- Received 5 September 2001; Accepted 20 March 2002 tractility, in electrophysiological properties, or in Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/jez.10122 r 2002 WILEY-LISS, INC.
adrenoceptors, if present, serve any functional role tory chemicals were from Merck (Darmstadt, amongst the lower vertebrates (Benfey, '82; Ask, '83). In addition, temperature-dependent effects ofadrenaline and other sympathomimetics have been reported for the frog myocardium, suggesting Male frogs (Rana ridibunda) weighing 100–120 a conversion of the adrenoceptor form from a b- g were caught in the vicinity of Thessaloniki, type at high to a a-type at low temperatures Greece, and were supplied by a local dealer. They (Kunos and Nickerson, '76; Chiu and Chu, '89).
were kept in containers in fresh water at 201C However, this has been questioned in a more under a 12-hr light/12-hr dark photoperiod.
recent study (Herman et al., '96).
Animals were not fed and were used a week after a1-Adrenergic receptors belong to the larger arrival. Care of the animals was according to the family of Gq/11-protein-coupled receptors that Guidelines for the Care and Use of Laboratory initiate signals by activating phospholipase C- Animals published by the Greek Government dependent hydrolysis of membrane phosphoinosi- (160/1991), based on European Union regulations tides, thus leading to production of Ins(1,4,5)P3 and diacylglycerol. The former regulates intracel-lular Ca2þ movements into a variety of mamma- Membrane preparation lian tissues, whereas the latter is the physiologicalactivator of protein kinase C (Berridge, '93; Zhong Frogs were decapitated, hearts were immedi- and Minneman, '99). Other signaling pathways ately excised, and ventricles were homogenized at have also been shown to be activated by a 41C in 9 v/w of buffer A (50 mM Tris, 100 mM adrenergic receptors, among which is Ca2þ influx NaCl and 2 mM EDTA, adjusted to pH 7.4 with HCl) using an Ultra-turrax homogenizer. Several Ca2þ channels, arachidonic acid release, and hearts were used (approx 2 g) for each membrane phospholipase D activation; however, these may preparation. Homogenates were centrifuged at vary depending on the cell type (Suzuki et al., '90; 20,000 g for 20 min at 41C. Pellets were dispersed Xing and Insel, '96; Noguera et al., '97; Ruan et al., in the same volume of buffer B (50 mM Tris and '98; Zhang et al., '98). In rat tissues, molecular 1 mM EDTA adjusted to pH 7.4) as used for the cloning and pharmacological studies have revealed initial homogenization using a ground glass the existence of three a homogenizer. The centrifugation and resuspen- subtypes, namely, a sion steps were repeated two more times. After a 1A, a1B, and a1D (Stewart et al., '94; Graham et al., '96). However, the existence final centrifugation, the pellets were resuspended in buffer B at the appropriate tissue concentration 1-adrenoceptors in the heart of lower verte- brates has not been clearly demonstrated, and (0.1 g original tissue weight per milliliter) and there has been no evidence for their coupling to kept at 701C until use. Thawed suspensions signal transduction pathways.
were rehomogenized using a ground glass homo- The aim of the present study was to directly genizer before use. Protein concentration was assess the expression of a determined by the method of Bradford ('76).
1-adrenoceptors in the frog heart by radioligand binding experiments and Radioligand binding furthermore to determine whether these receptorsare involved in the response of the heart to Radioligand binding using [3H]prazosin as the ligand was performed as previously described(Lazou et al., '94). Briefly, aliquots of the mem- MATERIALS AND METHODS brane suspensions (100 ml) were incubated with[3H]prazosin in a total volume of 1 ml buffer B at 251C for 60 min in the presence or absence of [3H]prasozin (specific activity 79 Ci/mmol) was competing drugs. The incubation was terminated from New England Nuclear (Boston, MA), and by rapid vacuum filtration over Whatman GF/C myo-[3H]inositol (specific activity 18 Ci/mmol) was filters, and each filter was washed with 15 ml ice- from Amersham Pharmacia Biotech (Merck Hel- cold buffer B. Filters were immersed in 1 ml of las, Glyfada, Greece). GF/C filters were from double-distilled water in scintillation vials and Whatman (Kent, UK). Adrenaline, noradrenaline, counted in Fluoran HV (BDH) in a LKB/Wallac phenylephrine, and prazosin were from Sigma- (Amersham Pharmacia Biotech, Merck, Hellas, Aldrich (Deisenhofen, Germany). General labora- Greece) scintillation counter. Nonspecific binding a1-ADRENOCEPTORS AND PI HYDROLYSIS IN FROG HEART was determined in the presence of 20 mM prazosin.
[3H]inositol phosphates were separated by a To determine the affinity (Kd) and the maximal method based on that of Berridge et al. ('83). The binding capacity (Bmax) of [3H]prazosin to cardiac entire neutralized cell extract was chromato- a1-adrenoceptors, saturation curves were con- graphed on a 0.5-  2.5-cm column of Bio-Rad structed by incubating membranes with increas- AG1x8 formate form (100–200 mesh) equilibrated ing concentrations of [3H]prazosin (0.05–18 nM) with water. Each column was washed with water and the data were analyzed by the method of (2 ml), 5 mM disodium tetraborate/60 mM sodium Scatchard. Competition binding experiments were formate (3 ml), and finally with 3 ml 0.1 M formic carried out at 0.8 nM [3H]prazosin with increasing acid/1.0 M ammonium formate; the last wash was concentrations of agonists.
retained. Fifteen milliliters of scintillation fluidwas added to the final wash. Radioactivity was Heart perfusions and prelabeling counted on a liquid scintillation counter.
of phosphoinositide (PI) pools After rapid excision, hearts were immersed in ice-cold saline and then mounted on a two-way The results are presented as means 7 SEM of n recirculating Langendorff perfusion apparatus.
experiments. Curve fitting was performed using The heart and the perfusion fluids were kept in the Prism program (GraphPad Software, San Diego, CA). Saturation binding experiments were standard perfusion medium was amphibian Ring- analyzed by fitting rectangular hyperbolic func- er containing (in mM): NaCl 103, CaCl2 1.8, tions to the experimental data to determine the NaHCO3 23.8, Na2HPO4 0.6, KCl 2.5, MgCl2 1.8, number of binding sites (Bmax) and their affinity and glucose, pH 7.4. The perfusion fluid was for the radioligand (Kd). Competition binding data equilibrated with a gas mixture containing 95% were analyzed using either one- or two-site O2/5% CO2, and perfusion pressure was main- models. A two-site fit was accepted only if it was tained constant at 45 cm H2O (4.41 kPa).
statistically better than a one-site model as In all experiments, a 15-min equilibration assessed by the use of the F-test (P o 0.05). IC50 period was allowed during which hearts were values from competition experiments were con- perfused with the standard perfusion medium.
verted to Ki values using the Cheng-Prusoff This was followed by a 2-hr perfusion with equation (Cheng and Prusoff, '73) after ensuring medium containing myo-[3H]inositol and finally a that the Hill coefficient (nH) was not different 5-min perfusion with standard perfusion medium.
from unity. Concentration-response data were At the end of this period, LiCl was added to the fitted by nonlinear regression to a four-parameter perfusion medium to a final concentration of 10 logistic equation. The Instat program (Graphpad mM, and the perfusion was continued for another Software) was used for all statistical calculations 10 min before switching to a perfusion medium and P o 0.05 was considered significant.
containing 10 mM LiCl and adrenaline (0.3–300mM). Perfusion with this medium was continued Characterization of a1-adrenoceptor Measurement of PI hydrolysis binding in membrane fractions from heart At the end of the perfusion period, hearts still Binding experiments were carried out using attached to the perfusing system were quickly [3H]prazosin, commonly classified as a specific a1- frozen between aluminum tongs cooled to the adrenergic receptor antagonist. Preliminary ex- temperature of liquid N2. Frozen perfusate as well periments indicated that [3H]prazosin bound to as connective tissue was removed and the frozen membranes prepared from frog heart in a satur- muscle was immediately homogenized in 5 vol wt.
able and reversible mode. Specific binding was of 0.8 M HClO4. Following centrifugation, the linearly dependent on tissue concentration (2–15 extract was neutralized to pH 7.0 with 0.8 M KOH mg of original heart wt) and reached a plateau at after 1M Tris base was added to a final concentra- 45 min (results not shown).
tion of 10 mM. Precipitated KClO4 was removed The number and affinity of a1-adrenergic bind- by centrifugation and the supernatant fraction ing sites were assessed by incubating heart was used for the separation of [3H]inositol membranes with [3H]prazosin ranging from 0.05 to 18 nM (Fig. 1). Scatchard plots for [3H]prazosin A. LAZOU ET AL.
ment experiments were performed in the presenceof 0.8 nM [3H]prazosin and increasing concentra-tions of unlabeled prazosin (Fig. 2). Nonlinearregression analysis of competition curves gave aKd of 6.19 7 1.9 nM and a Bmax of 15.61 7 5.96fmol/mg original wet weight. These results are inclose agreement with the values obtained from thesaturation binding experiments.
The binding of [3H]prazosin to heart mem- branes in the presence of various adrenergicagonists was also investigated. Adrenaline, nora-drenaline, and phenylephrine, in the presence ofDL-propranolol, [3H]prazosin. Competition curves were monocom-ponent (Fig. 3). Values of pKi for adrenaline, Saturation curves of [3H]-prazosin binding to noradrenaline, and phenylephrine were 4.97 7 purified membranes from frog heart. Membranes (100 ml 0.20, 5.09 7 0.04, and 4.96 7 0.14, respectively, containing 10 mg of original tissue weight) were incubated at and nH values were as follows: adrenaline 1.04 7 251C for 60 min with increasing concentrations of [3H]prazo-sin ranging from 0.05 to 10 nM. Total (,) and nonspecific 0.11, noradrenaline 0.94 7 0.06, and phenylephr- binding (&) were determined in the absence and presence of ine 0.98 7 0.21 (Table 1). These results show that 20 mM prazosin (*). Specific binding was calculated by the affinities of each individual agonist for a1- subtracting nonspecific from total binding. The best fit was adrenoceptor subtypes are equal.
determined with a one-site model. The plot is a representativeof four similar experiments run in duplicate.
Stimulation of PI hydrolysis by adrenaline binding were linear, indicating a homogeneous class of binding sites. Analysis of saturation curves The effect of adrenaline on the production of by nonlinear regression revealed an equilibrium [3H]inositol phosphates in the frog heart prela- binding constant (Kd) of 4.19 7 0.56 nM and a beled with [3H]inositol was studied. The experi- mean receptor density (Bmax) of 14.66 7 1.61 ments were carried out both in the absence and fmol/mg original wet weight. To further evaluate presence of propranol to block the b-adrenergic the specificity of [3H]prazosin binding, displace- activity of adrenaline. In the presence of maxi-mally effective concentrations of adrenaline (100mM), PI hydrolysis in frog heart was linear withtime (results not shown). Adrenaline stimulatedPI hydrolysis in a concentration-dependent man-ner. The data from individual experiments used tocompile the composite curve in Fig. 4 were fitted toa sigmoid curve, and the derived data are shown inTable 2. In the absence of propranolol, adrenaline TABLE 1. Binding parameters derived from the competition by adrenaline, noradrenaline, phenylephrine, and prazosin for [3H]prazosin binding sites in membranes from frog heart Homologous competition curve for prazosin. Mem- brane fractions of heart were prepared and incubated with increasing concentrations of prazosin in the presence of 0.8 nM [3H]prazosin. [3H]prazosin binding is expressed as a Note: Competition curves were performed as described in Materials and percentage of total binding in the absence of prazosin. All Methods. Data were best ¢tted to either a one-site or two-site model. Values values represent the mean 7 SEM of five different experi- are the means7SEM from three to ¢ve separate determinations using di¡er- ments. Where no error bars are shown, their size is smaller ent membrane preparations. pKi is the negative log of Ki; nH is the Hill coe⁄- than the size of the symbol.
a1-ADRENOCEPTORS AND PI HYDROLYSIS IN FROG HEART Competition between [3H]prazosin and (A) adrenaline, (B) noradrenaline, and (C) phenylephrine for binding to membrane preparations of frog heart. Membrane fractions of heart were prepared and incubated with various concentrations ofagonists in the presence of 0.8 nM [3H]prazosin as described in Materials and Methods. [3H]prazosin binding is expressed as apercentage of binding in the absence of agonists following subtraction of blanks. A one-site curve was significantly better than atwo-site curve (P o 0.001). Data are the mean 7 SEM of three to four separate membrane preparations where assays wereperformed in duplicate. Where no error bars are shown, their size is smaller than the size of the symbol.
stimulated PI hydrolysis by 8.5-fold over the basal rate with an EC50 value of about 31 mM. The mediated through these receptors in the isolated presence of propranolol did not affect the maximal perfused heart. To our knowledge, this is the first stimulation; however, it produced a small shift of direct evidence supporting the presence of func- the curve to the left that was not significant, and tional a1-adrenergic receptors in the heart of a the computed EC50 was 21 mM. The EC50 value for lower vertebrate.
the stimulation of PI hydrolysis closely resembles Cardiac cells from amphibian species have been the affinity constant for adrenaline observed in widely used as model systems for the study of the the radioligand binding, indicating that the cel- electrophysiological properties of the heart, and lular action was mediated by the same receptor the role of b- adrenergic receptors has been clearly identified in the binding studies.
described. However, few studies have been carriedout on the presence and the functional role of a- adrenergic receptors. The participation of thesereceptors in the positive inotropic effect of cate- The present study confirms the presence of a cholamines in the amphibian heart has been adrenoceptors in the heart of amphibians and questioned (Morad et al., '78; Soustre and Rako- furthermore provides clear evidence for an adre- tonirina, '81). In the frog heart, Chiu and Chu('89) reported that at low temperatures a catecho-lamine effect was exerted by a-adrenoceptors withthe b-adrenoceptor population being scarce oreven absent at this temperature. In contrast,Herman et al. ('96) showed that a1-, a2-, and b-adrenergic receptors coexist in the heart of theAmerican bullfrog R. catesbeiana, and they re-ported no change in b-adrenergic receptors in TABLE 2. Stimulation of phosphoinositide hydrolysis by adrenaline in the perfused frog heart Extrapolated maximal Stimulation of phosphoinositide hydrolysis by adrenaline. Hearts were prelabeled with [3H]inositol as described in Materials and Methods and then perfused with Note: Experiments were performed and data were ¢tted to sigmoid curves as various concentrations of adrenaline in the absence (*) or described in Material and Methods. From sigmoid curves, extrapolated max- presence (&) of 20-fold molar excess of DL-propranolol for 45 imal rate of phospho[3H]inositide (PI) hydrolysis was expressed relative to min. [3H]inositol phosphates were isolated and data fitted to derived basal rate of hydrolysis data to give extrapolated maximal response.
sigmoid curves. Each data point is the mean 7 SEM of five to pEC50, log of half maximum e¡ective concentration.When present, DL-pro- eight different experiments.
pranolol was in 20 -fold molar excess relative to adrenaline concentrations.
warm-acclimated frogs and in a-adrenergic recep- production of inositol phosphates stimulated by tors in cold-acclimated ones. However, there is no adrenaline in the perfused frog heart. Increasing detailed characterization of a1-adrenoceptors and concentrations of adrenaline resulted in a classic their functional role in the heart of lower dose-response increase in phosphoinositide hydro- vertebrate species.
lysis (Fig. 4). Maximal stimulation was about 8.5- To reexamine the possibility that a1-adrenergic fold over control, which is comparable to that receptors are actually present in the frog heart, observed in the rat heart (Table 2, Lazou et al., binding studies were carried out using membranes '94). However, the EC50 of 31 mM (Table 2) is prepared from heart. Saturation experiments much lower (about two orders of magnitude) than using [3H]prazosin and competition experiments that reported for rat heart. The EC50 value for using a1-adrenoceptor agonists and selective an- adrenaline was slightly decreased (21 mM) in the tagonists were undertaken. Analysis of the data presence of propranolol. This is presumably partly obtained in the present study suggested a single attributable to inhibition of binding of adrenaline class of binding sites for [3H]prazosin displaying a to b-adrenoceptors. The pEC50 value for the Kd of about 4.19 nM (Fig. 1). This value is in close stimulation of PI hydrolysis by adrenaline corre- agreement to that obtained in competition experi- lates well with the corresponding pKi value for ments where the potency of prazosin in inhibiting this agonist in [3H]prazosin binding competition 50% of tracer binding was calculated to be about assays (Tables 1 and 2). This supports close 6.19 nM (Fig. 2, Table 1). Since this is the first coupling between a1-adrenoceptor occupation and detailed study for the characterization of a1- phosphoinositide hydrolysis and suggests that the adrenergic receptors in the heart of a lower cellular action was mediated by the same receptor vertebrate species, these values can only be identified in the binding studies.
compared to those reported for mammalian heart.
The fact that a1-adrenoceptors are coupled to It can be noted that the affinity of a1-adrenocep- phosphoinositide hydrolysis in frog heart suggests tors for prazosin is two orders of magnitude lower that these receptors may participate in signaling in the frog heart than in the rat heart, where Kd events that determine cardiac physiology. It was reported to range from 25 to 170 pM (Hanft remains to be established whether these receptors and Gross, '89; Lazou et al., '94; Seraskeris et al., contribute to signal transduction leading to cell 2001). In a study in the bullfrog heart ventricle growth and gene expression, as in other cell and atrium, the authors were unable to determine systems (Spector et al., '97; Lazou et al., '98; the Kd because of the very low number of binding Williams et al., '98). If this also happens in the frog sites present (Herman et al., '96). As to the heart, apart from the evolutionary importance, maximal binding density, the value obtained in this system may provide a useful tool for studying this study, 14.66 fmol/mg original wet weight., signaling events.
markedly differs from that reported by Herman et In summary, our data clearly indicate that a1- al. ('96) in the American bullfrog heart. Apart adrenoceptors are present in frog cardiac cells and from the fact that different experimental protocols are coupled to phosphoinositide hydrolysis and the have been followed to determine nonspecific production of inositol phosphates, which is stimu- binding in the two studies, these differences may lated by adrenaline in the perfused heart.
reflect interspecies variability in cell surface a1-adrenoceptor density.
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The Journal of Neuroscience, December 16, 2009 • 29(50):15675–15683 • 15675 Frontal Feedback-Related Potentials in Nonhuman Primates:Modulation during Learning and under Haloperidol Julien Vezoli1,2 and Emmanuel Procyk1,21Inserm, U846, Stem Cell and Brain Research Institute, 69500 Bron, France, and 2Universite´ de Lyon, Lyon 1, UMR-S 846, 69003 Lyon, France

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