Introduction

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The sympathetic nervous system plays an important role in regulating the heart, including contraction, gene expression, hypertrophy, and apoptosis. Catecholamines bind to adrenoceptors localized on the outer surface of myocardial cell membranes to trigger these effects. ₁-Adrenoceptors exist on mammalian myocardial cells (Benfey, 1990; Li et al., 1997) and their activation produces a positive inotropic response (Li et al., 1997; Otani and Das, 1994). The stimulation of cardiac ₁-adrenoceptors also increases protein synthesis and induces cardiac hypertrophy (Simpson et al., 1982; Milano et al., 1994; Ramirez et al., 1997). In addition, cardiac ₁-adrenoceptors regulate the -adrenoceptor-mediated positive inotropic response (Osnes et al., 1989; Barrett et al., 1993; Zhang et al., 1994).

₁-Adrenoceptors were originally subdivided pharmacologically into two subtypes termed _1A and _1B. The _1A subtype has a high affinity for the selective antagonists 2-(2,6-dimethoxphenoxyethyl)-aminomethyl-1,4 benzodioxane (WB4101), 5-methyl-urapidil (5-MU), and (+)niguldipine, and is insensitive to inactivation by the alkylating agent chlorethylclonidine (CEC). The _1B subtype has a low affinity for these competitive antagonists and is completely inactivated by CEC (Han et al., 1988; Gross et al., 1988; Boer et al., 1989; Morrow and Creese, 1986). Based on this scheme, using functional and radioligand binding methods, Han et al. (1991; Yu and Han, 1994) demonstrated that rat atria contains both _1A and _1B subtypes in a ratio of 1:2 and both of two subtypes are involved in the positive inotropic response. Others (Michel et al., 1994; Lazou et al., 1994) also reported that adult rat heart contains _1A- and _1B-adrenoceptors in an approximately 20 to 25:80 to 75 ratio. Both _1A- and _1B-adrenoceptor subtypes have been suggested to play a role in ₁-adrenoceptor-mediated positive inotropic responses and increasing phosphoinositide hydrolysis in rat and rabbit hearts (Endoh et al., 1992; Yu and Han, 1994; Michel et al., 1994; Lazou et al., 1994; Nagashima et al., 1996). In rat hearts, this involved primarily the _1B, and in rabbit hearts the _1A (Deng et al., 1996b). However, the Muramatsu group (Noguchi et al., 1993, 1995) presented evidence for the presence of at least three distinct ₁-adrenoceptor subtypes, _1A, _1B, and _1L, in rat hearts, and suggested that ₁-adrenoceptor-mediated positive inotropic response could not be explained by the _1A and _1B subclassification.

Molecular cloning and pharmacological studies have made it clear that there are actually three ₁-adrenoceptors subtypes-_1A-, _1B-, and _1D (IUPHAR Committee of Receptor Nomenclature and Drug Classification, 1995). The distribution of ₁-adrenoceptor subtype mRNA in human and rat hearts has been extensively studied using RNase protection assays (Price et al., 1994; Rokosh et al., 1994) or reverse transcription-polymerase chain reaction (Faure et al., 1995). Those studies demonstrated that all three subtypes are expressed in human and rat hearts. However, the proportion of ₁-adrenoceptor subtype protein in rat heart, and the inotropic responses mediated by these different subtypes have not yet been established. In the present study, we characterized the distribution of the three ₁-adrenoceptor subtypes by radioligand binding assays (RBA) and RNase protection assay, and also determined the role of each subtype in mediating the inotropic response in rat heart.

	Experimental Procedures

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Materials

The drugs used were obtained from the following sources: WB4101, (+)-niguldipine, 5-MU, 8-[2-[4[-(2-methoxyphenly)-1-piperazinyl]-8-azaspiro]4,5]decane-7,9-dione dihydrochloride (BMY7378), CEC dihydrochloride, and spiperone (Research Biochemicals Inc., Natick, MA); 2-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone (BE2254; Beiersdorf, Hamburg, Germany); noradrenaline, phentolamine, yohimbine, desmethylimipramine, normetanephrine, (±)-propranolol, and phenoxybenzamine (POB; Sigma, St. Louis, MO); N-[2-2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-,-dimethyl-1H-indole-3-ethanamine hydrochloride (RS-17053; Roche Bioscience); ¹²⁵I-Na⁺ (Beijing Institute of Atomic Energy, Chinese Academy of Science, Beijing, China); RNase protection assay set (Promega Inc., Beijing); and [-³²P]Utriphosphate ([-³²P]UTP, Huirui Inc. Beijing, China).

Membrane Preparation

Human embryonic kidney (HEK)293 cells were harvested by scraping confluent 75-cm² flasks and pelleted by centrifugation at 500g for 5 min, washed with 10 ml physiological buffer solution (Na₂HPO₄, 20 mM; NaCl, 154 mM, pH 7.6, PBS), and centrifuged again. Cells were homogenized with a Polytron (speed 6, 10 s) in 10 ml of PBS. Membranes were collected by centrifugation at 20,000g for 10 min and resuspended in PBS (one flask HEK293 cells transfected with _1A or _1D per 5-ml final suspension, or one flask HEK293 cells transfected with _1B per 20-ml final suspension).

Rats were sacrificed by cervical dislocation and the hearts were removed. Crude particulate fractions of ventricles and atria were made as described previously (Han et al., 1991). Briefly, tissue was homogenized with a Polytron in 20 ml of PBS, centrifuged at 20,000g for 10 min, resuspended in PBS, and centrifuged again. Membranes were then resuspended in PBS to the appropriate tissue concentration (about 1 mg protein/ml).

RBA

BE2254 was radioiodinated to theoretical specific activity of 2200 Ci/mmol as described by Engel and Hoyer (1981) and stored at 20°C in methanol. Measurement of specific ¹²⁵I-BE2254 binding was performed by incubating membrane preparations with ¹²⁵I-BE2254 in PBS in a final volume of 250 µl for 20 min at 37°C in the presence or absence of competing drugs. After 20 min, the incubation was terminated by adding 10 ml of 10 mM Tris-HCl (pH 7.4) and the mixture was filtered over a glass fiber filter (Type 49 25, Hong Guang Paper Manufactory, Shanghai) in a vacuum. Each filter was washed with 10 ml of 10 mM Tris-HCl (pH 7.4), dried, and its radioactivity (dpm) was measured. Nonspecific binding was determined in the presence of 10 µM phentolamine, and was less than 15%. Protein concentrations of the preparation were measured by the Coomassie Brilliant Blue method.

To determine the affinity (K_D) and the maximal binding capacity (B_max) of ¹²⁵I-BE2254 to cardiac ₁-adrenoceptors, saturation curves were determined by incubating tissues with increasing concentrations of ¹²⁵I-BE2254 (15-520 pM, 18,750-625,000 dpm) and the data were analyzed by the method of Scatchard. To protect _1A- or _1D-adrenoceptor from POB alkylation, the membrane preparations were preincubated with 5-MU (0.3 µM) or BMY7378 (0.5 µM) for 20 min at 37°C and then 0.3 µM POB was added for an additional 30-min incubation, followed by three washes with PBS. Saturation curves were obtained as described above. To determine the affinity of BMY7378, (+)-niguldipine, RS-17053, spiperone, 5-MU, and WB4101 to cloned and cardiac ₁-adrenoceptors, their potencies competing for specific ¹²⁵I-BE2254 binding sites were determined by incubation of one concentration of ¹²⁵I-BE2254 (50-55 pM, around 62,500 dpm) in the presence or absence of 16 concentrations of the antagonists. The best two-site fit for a competitive binding curve was calculated by minimizing the sum of squares of the errors using nonlinear regression analysis for heart membrane preparations. Two-site models were compared with one-site models to determine whether the increase of goodness of fit was significantly more than would be expected on the basis of chance alone using a partial F test. If a two-site model was confirmed, IC₅₀ values for each binding site were determined by nonlinear regression analysis. For cells transfected with cloned ₁-adrenoceptors, IC₅₀ values were determined by nonlinear regression analysis. K_i values were calculated by the method of Cheng and Prusoff (1973).

After the protection protocol, competitive binding curve for 5-MU and BMY7378 were performed as described above.

Inotropic Responses

Rats were sacrificed by cervical dislocation, the hearts were exposed, and the left atria were rapidly removed. Inotropic responses of left atria were measured as described previously (Yu and Han, 1994). Briefly, tissues were placed in Krebs' solution (composition in mM: NaCl, 120; KCl, 5.5; CaCl₂, 2.5; NaH₂PO₄, 1.2; MgCl₂, 1.2; NaHCO₃, 20; dextrose, 11; and Na₂EDTA, 0.029) equilibrated with 95% O₂/5% CO₂, and maintained at 37°C in an organ bath with a volume of 10 ml, containing 30 µM propranolol to block the -adrenoceptor response, 0.1 µM yohimbine to block the ₂-adrenoceptor response, and 0.1 µM desmethylimipramine and 1 µM normetanephrine to block the uptake of noradrenaline by nerve endings and cardiac tissue. The tissues were attached to force-displacement transducers for measurement of isometric tension and stimulated by electrical pacing (1 Hz, 5 ms, 2 times threshold voltage). A resting tension of 0.5g was applied to all of the preparations. A cumulative concentration-contractile response curve for noradrenaline was then obtained. The potency of noradrenaline was expressed as pD₂, which was the negative logarithm to base 10 of the EC₅₀ (the dose that produces 50% of the maximal response to the drug).

In experiments examining the effect of CEC, cumulative concentration-response curves for noradrenaline (0.01-300 µM) were generated first. After washing and 30-min equilibration, preparations were incubated with or without 30 µM CEC for 30 min. Preparations were then washed and equilibrated for another 30 min, and cumulative concentration-response curves for noradrenaline were repeated.

An attempt was made to protect _1A or _1D subtypes from POB alkylation by use of a protocol similar to that used by Dunn et al. (1991). Tissues were exposed to 5-MU (0.3 µM) or BMY7378 (0.5 µM) 20 min before POB (0.3 µM), which was left in contact with the tissues for another 30 min. The tissues were then washed seven times over a 45-min period. After that, cumulative concentration-response curves for noradrenaline were performed.

We determined pA₂ values for prazosin, yohimbine, 5-MU, RS-17053, BMY7378, WB4101, and spiperone by the method of Arunlakshana and Schild (1959). After control noradrenaline concentration-response curves were made, the preparations were incubated with Krebs' solution with antagonist (three different concentrations with 0.5 log M increments) or vehicle for 30 min. A second curve for noradrenaline was then made in the presence of antagonist or vehicle (time control). EC₅₀ was calculated by nonlinear regression. The pA₂ and slope for the antagonist were determined from a Schild plot.

RNase Protection Assays

Isolation of Total RNA. Frozen heart tissues were ground into a powder in liquid nitrogen, and homogenized with a high-speed homogenizer. Total RNA was then extracted by the single step method. RNA samples were then quantified using a spectrophotometer at 260/280 nm, and aliquoted into 30 µg samples, which were stored at 70°C for later use.

RNA Probe Labeling. Antisense RNA probes were transcribed with T₃ or T₇ RNA polymerase from DNA templates in the presence of [-³²P]UTP and purified on an 8 M urea-6% polyacrylamide gel before use. The DNA templates for antisense RNA synthesis were as follows: a 487-base pair (bp) (HindIII/XhoI) fragment of _1A cDNA; a 306-bp (BamHI/PstI) fragment of _1B cDNA; and a 414-bp (EcoRV/SmaI) fragment of _1D cDNA. The above fragments were cloned into pBluescript SK (). Sizes of the probes/protected fragments were as follows: _1A, 572/487; _1B, 356/306; _1D, 459/414.

RNase Protection Assays. RNase protection assays were conducted as described and validated previously (Xu and Han, 1996). Forty micrograms of total RNA was used to hybridize with probes specific for each of the three rat ₁-adrenoceptor mRNAs. Forty micrograms of yeast RNA was used to control. The autoradiographic bands were quantified by an Imaging Analysis System (Q550 IW; Leica, Wetzlar, Germany).

Animal Care

Experiments were performed with male, 180- to 200-g, hooded Wistar rats. The rats were housed in groups at room temperature (22-25°C), and were provided with a conventional diet and water ad libitum. Experiments were approved by the Committee on the Ethical Aspects of Research Involving Animals of the Beijing Medical University.

Statistics

The results shown in the text and tables are expressed as means ± S.E. Statistical analysis of the data was done by paired t test or Student's t test. Two groups of data were considered to be significantly different when p < .05.

	Results

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Expression of ₁-Adrenoceptor Subtypes in Rat Ventricle and Left Atrium

Scatchard analyses for the saturation curves of ¹²⁵I-BE2254 binding to the crude membrane preparations yielded a B_max of 119 ± 11 fmol/mg protein and a K_D of 34 ± 0.4 pM in ventricle (n = 5). After preincubation of the preparations with 20 µM CEC, the B_max decreased by 72% (33 ± 6 fmol/mg protein, p < .01) whereas K_D values did not change significantly (36 ± 0.3 pM, Fig. 1). In left atrium, B_max values were 119 ± 15 fmol/mg protein and K_D values were 85 ± 29 pM (n = 5). Nether B_max nor K_D values were significantly different between ventricle and left atrium.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of CEC on 1-adrenoceptor density in rat ventricle. The saturation experiments were performed using 125I-BE2254 as radioligand, and Bmax values were calculated by Scatchard analysis. : control; : pretreated with 30 µM CEC.

Specific ¹²⁵I-BE2254 binding was competitively inhibited by (+)-niguldipine, RS-17053, BMY7378, 5-MU, WB4101, and spiperone in a concentration-dependent manner (Fig. 2). The inhibition curves from both ventricle and left atria were relatively shallow, with Hill coefficients significantly less than unity. Computer analyses showed that the curves were better fit by a two-site than a one-site model (p < .01). The percentages of high-affinity sites for these antagonists were not significantly different between ventricle and left atrium (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Inhibition of 125I-BE2254 specific binding to membranes of rat hearts by subtype-selective antagonists. The best two-site fit was calculated by nonlinear regression analysis using GraphPad Prism.

View this table: [in this window] [in a new window]   TABLE 1 Inhibition by 1-adrenoceptor antagonists of 125I-BE2254 specific binding to membranes of rat ventricle and atrium

After preincubation of the rat heart membranes with 5-MU and POB (n = 4) or BMY7378 and POB (n = 6), B_max values were decreased by 59 and 70%, respectively. K_D values did not change significantly (Table 2). The inhibition curves from these preparations were relatively steep, and the Hill coefficients were not significantly less than unity. Computer analyses showed that the curves were better fit by a one-site than a two-site model (p < .01). The pK_i values were 8.12 ± 0.28 for 5-MU and 8.3 ± 0.4 for BMY7378 (Fig. 3), respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of the protection protocol on inhibition of 125I-BE2254 specific binding to membranes of rat heart by selective antagonists. Protection protocol was described in Experimental Procedures. Briefly, the preparations were preincubated with 0.3 µM 5-MU () or 0.5 µM BMY7378 () for 20 min and then 0.3 µM POB was added for an additional 30-min incubation, followed by three washes with PBS.

Binding Affinities (pK_i ) for ₁-Adrenoceptor Subtype-Selective Antagonists at Cloned ₁-Adrenoceptor Subtypes

The ₁-adrenoceptor antagonist prazosin, ₂-adrenoceptor antagonist yohimbine, and ₁-adrenoceptor subtype-selective antagonists, RS-17053, BMY7378, 5-MU, WB4101, and spiperone inhibited binding of ¹²⁵I-BE2254 to _1A-, _1B-, and _1D-adrenoceptors stably expressed in HEK293 cells in a concentration-dependent manner. Their K_i values were shown in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Comparison of the functional potencies of 1-adrenoceptor subtype-selective antagonists to inhibit noradrenaline-induced positive inotropic responses in rat left atria to the radioligand binding affinities at cloned 1-adrenoceptor subtypes expressed in HEK293 cells

Role of ₁-Adrenoceptor Subtypes in Positive Inotropic Response in Electrically Driven Isolated Left Atria

Effect of CEC on the Positive Inotropic Effect of Noradrenaline. In the presence of 30 µM propranolol, which did not change basal tension significantly, noradrenaline produced a positive inotropic response with a pD₂ value of 4.35 ± 0.08 and maximal tension increment of 255 ± 58 mg in isolated electrically driven left atria. Pretreatment of preparations with 30 µM CEC did not change basal contractile tension, but shifted the concentration-response curve for noradrenaline to the right and downward (Fig. 4). The pD₂ value was decreased to 3.68 ± 0.11 (p < .05) and maximal tension increment was reduced by 73% (68 ± 38 mg) compared with the control.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of CEC on noradrenaline-induced positive inotropic responses in rat left atria (presence of 30 µM propranolol). : control; : pretreated with 30 µM CEC.

Effects of Protection Protocol on Noradrenaline-Induced Positive Inotropic Response in Rat Left Atria. Preincubation with POB (0.3 µM) alone virtually abolished responses to noradrenaline in left atrial preparations (data not shown). Preincubating with 5-MU (0.3 µM) to protect _1A-adrenoceptors and then preincubating with POB (0.3 µM) to inactivate the other subtypes reduced the maximum response to noradrenaline by 43% of control. Combining preincubation with BMY7378 (0.3 µM) and POB (0.3 µM), the maximum response to noradrenaline was reduced by 79% (Fig. 5, Table 4).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of the protection protocol on noradrenaline-induced positive inotropic response in rat left atria (presence of 30 µM propranolol). Protection protocol was described in Experimental Procedures. The preparations were preincubated with 0.3 µM 5-MU () or 0.5 µM BMY7378 () for 20 min, 0.3 µM POB was added for an additional 30-min incubation, and then the tissues were washed seven times over a 45-min period.

pA₂ Values for Prazosin and Yohimbine in Antagonizing Noradrenaline-Induced Positive Inotropic Response. Prazosin (3-30 nM in the absence of yohimbine) or yohimbine (1-10 µM) competitively antagonized the noradrenaline-induced positive inotropic response with pA₂ values of 9.86 ± 0.09 and 7.00 ± 0.08, and a slope in the Schild plot of 1.2 ± 0.2 and 1.1 ± 0.2, respectively.

pA₂ Values for RS-17053, 5-MU, BMY7378, WB4101, and Spiperone for Antagonizing Noradrenaline-Induced Positive Inotropic Response. RS-17053, 5-MU, BMY7378, WB4101, and spiperone inhibited the noradrenaline-induced positive inotropic response in a competitive and concentration-dependent manner. pA₂ values for RS-17053, 5-MU, BMY7378, WB4101, and spiperone were determined from a Schild plot and were 8.39, 8.25, 6.80, 8.42, and 7.97, respectively, and the slopes were significantly less than unity (p < .05, respectively; Table 3).

Comparison between the Functional Potencies (pA₂) of ₁-Adrenoceptor Subtype-Selective Antagonists and their Binding Affinities (pK_i) at Cloned ₁-Adrenoceptor Subtypes. The potencies (pA₂) for the ₁-adrenoceptor subtype-selective antagonists for inhibiting contractile responses to noradrenaline in rat left atria correlated well with binding pK_i values at cloned _1A and _1B subtypes (_1A, r² = 0.73, p < .05; _1B, r² = 0.66, p < .05; Fig. 6). However, the functional pA₂ values correlated poorly with binding pK_i values at the cloned _1D subtype (r² = 0.35, p > .05; Fig. 6).

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Correlation between the functional potencies (pA2) of 1-adrenoceptor subtype-selective antagonists to inhibit noradrenaline-induced positive inotropic responses in rat left atria and their radioligand binding affinities (Ki) at cloned 1-adrenceptor subtypes expressed in HEK293 cells. Correlation constants (r2) were calculated by linear regression analysis using GraphPad Prism.

RNase Protection Assays

The mRNA levels for the three ₁-adrenoceptor subtypes were determined in rat hearts. A representative autoradiograph of an RNase protection assay is shown in Fig. 7. Quantification analysis showed that mRNAs for _1A, _1B, and _1D were 0.54 ± 0.14, 1.00 ± 0.04, and 1.00 ± 0.10 fmol, respectively, from 40 µg total RNA.

View larger version (109K): [in this window] [in a new window]   Fig. 7.   Representative autoradiograph of RNase protection assay in rat heart. Lanes 1 to 3: yeast RNA, negative control; lanes 4 to 6: mRNAs for 1A, 1B, and 1D, respectively.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

RBA are still the one of the most useful and effective methods for quantitating receptor expression in native tissues. However, accurate quantitation of closely related receptor subtypes requires highly subtype-selective antagonists. Because only moderately subtype-selective antagonists are available for the three closely related ₁-adrenoceptor subtypes, quantitation of subtypes coexisting in tissues is difficult. Although BMY7378 has a 100-fold higher affinity for cloned _1D- than for _1A- or _1B-adrenoceptors, it is still difficult to quantitatively separate these subtypes with RBA. We therefore used RBA and quantitation of mRNA to study ₁-adrenoceptor subtype expression in rat hearts, and used functional approaches to examine the role of each subtype in positive inotropic responses.

Total ₁adrenoceptor density was quantitated by saturation analysis of specific ¹²⁵IBE2254 binding, and the proportion of each subtype was determined by competition with _1A- or _1D-selective antagonists. These results were confirmed by selective inactivation of _1B- and _1D-adrenoceptors by CEC alkylation and protection from POB alkylation by subtype-selective antagonists.

We found similar total densities (B_max) of ₁-adrenoceptors in ventricle and left atrium, with similar affinities for ¹²⁵I-BE2254 (K_D)._. In ventricle, inhibition curves for _1A-selective antagonists were best fit to a two-site model, where the high-affinity site representing the _1A-adrenoceptor comprised 19 to 28% of total binding sites. This suggests that the _1A subtype may constitute about one-fourth of total ₁-adrenoceptors in rat heart. Similarly, BMY7378 was used to calculate the proportion of _1D-adrenoceptors. Displacement curves showed that about one-third of total ₁-adrenoceptor binding sites in ventricle had a high affinity for BMY7378. Using WB4101, which has a high affinity for _1A- and _1D-adrenoceptors but a low affinity for _1B-adrenoceptor, we found that high-affinity sites comprised 42% of total sites in ventricle. Conversely, spiperone is an _1B- selective antagonist, and high-affinity sites for spiperone were 42% of total in ventricle. These results suggest that the proportion of _1A-, _1B-, and _1D-adrenoceptors in rat ventricles are about 25, 45, and 30% respectively. There are no significant differences in the ratios of subtypes between ventricle and atrium.

CEC was originally suggested to preferentially alkylate the _1B and _1D subtypes. However, alkylation of different ₁-adrenoceptor subtypes by this compound is highly dependent on temperature, time, and drug concentration (Han and Minneman, 1995; Xiao and Jeffries, 1998), as well as subtype-specific differences in subcellular localization (Tsujimoto et al., 1998). In addition, it now seems clear that both _1B and _1D have similar sensitivities to CEC alkylation. _1A-Adrenoceptors can also be inactivated by CEC, but at a markedly slower rate than other two subtypes (Xiao and Jeffries, 1998). We found that after preincubating with 30 µM CEC the number of ¹²⁵I-BE2254 binding sites was reduced by 73% in heart. This result is consistent with the conclusion that the ratio of _1A to the combined _1B and _1D population was approximately 1:2.

Recently Yang et al. (1997) and Wolff and Scofield (1998) reported that _1D-adrenoceptors were not readily detectable in rat heart using RBA because BMY7378 showed steep and monophasic competition curves with a low affinity. This is contrary to the results of the present study, where substantial high-affinity binding of BMY7378 was observed in both ventricle and atrium. To further address the question of whether the _1D subtype exists in rat heart, we tried to isolate a homogeneous population of _1A- or _1D-adrenoceptors by the use of a receptor protection procedure. Receptors were protected with the _1A-selective antagonist 5-MU or the _1D-selective antagonist BMY7378 and subjected to alkylation with the nonsubtype-selective irreversible alkylating agent POB. In the absence of receptor protection, we found that POB (0.3 µM) completely abolished specific ¹²⁵I-BE2254 binding. However, when the _1A subtype was protected with 5-MU or the _1D subtype was protected with BMY7378, POB treatment reduced about one-third of ¹²⁵I-BE2254 binding sites of control, respectively. After this treatment, inhibition curves were relatively steep and better fit by a one-site model. These results provide more evidence that the proportion of _1A and _1D in rat heart are approximately 30 and 30%, respectively.

We also detected mRNA for all three ₁-adrenoceptor subtypes in rat heart. RNase protection assays showed that _1A, _1B, and _1D comprised 22, 39, and 39% of total ₁-adrenoceptor mRNA, respectively. These results are only slightly different from those obtained with RBA, and any differences between protein and mRNA may be due to differences in post-transcriptional regulation. Together, these data support the hypothesis that all three ₁-adrenoceptor subtypes coexist in rat heart.

Cardiac ₁-adrenoceptor responses to catecholamines occur in both normal heart and multiple pathophysiological states. Much of the physiological interest in ₁-adrenoceptor activation has focused on the nature, mechanisms, and consequences of the inotropic effects of these subtypes. Deng's group (1996a) determined possible ontogenic differences in the function of rat myocardial ₁-adrenoceptor subtypes. They showed that ₁-adrenoceptor agonists increased right ventricular contraction and phosphoinositide turnover primarily through _1A-adrenoceptors in neonatal heart, but both _1A- and _1B-adrenoceptors in adult heart. They also presented evidence that _1D-adrenoceptors play a minor role in adult rat heart (Deng et al., 1996b).

The coexistence of all three subtypes in rat heart makes it difficult to determine the role of each subtype. However, using several selective antagonists we used functional experiments to evaluate the role of each subtype in positive inotropic responses to noradrenaline in field-driven isolated left atria. The response to noradrenaline was totally blocked by 0.1 µM prazosin, showing that it was mediated through ₁-adrenoceptors. pA₂ values for subtype-selective antagonists in competitively inhibiting noradrenaline-induced contraction did not clearly correlate with their affinities for specific receptor subtypes. To examine this more, we performed RBA in subcloned HEK293 cells stably expressing _1A, _1B, and _1D-adrenoceptor to obtain more accurate K_i values for these compounds, and compared them to the pA₂ values obtained from the functional experiments. The results showed that the correlation was significantly higher at cloned _1A and _1B than at _1D-adrenoceptor subtypes, suggesting that positive inotropic responses to noradrenaline may be mediated by a combination of subtypes, and that the contribution of _1A and _1B may be stronger than _1D.

Functional experiments in left atria showed that the same concentration of CEC reduced the maximal response to noradrenaline by 73% similar to the decease in receptor binding. This supports the hypothesis that _1B and/or _1D subtypes play an important role in this response. To define the relative contributions of _1B and _1D subtypes, we also used a protection strategy. POB completely abolishes the response to noradrenaline in rabbit isolated saphenous artery (Daly et al., 1998) and rat isolated left atria in our present study. After protection of the _1A subtype with 5-MU, the response to the noradrenaline was 57% of control, suggesting that more than half of the noradrenaline-induced response was mediated by the _1A-adrenoceptor subtype. After protection of the _1D subtype with BMY7378, the maximum response to noradrenaline was only 21% of control, suggesting that _1D-adrenoceptors play a minor role in rat heart.

In summary, this study demonstrates the coexistence of the three ₁-adrenoceptor subtypes at the mRNA and protein levels in rat heart, and further elucidates the importance of both _1A- and _1B-adrenoceptors in the positive inotropic response to noradrenaline. The functional importance of _1D-adrenoceptors in rat heart remains unclear.