Introduction

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The chain of events from drug administration to a certain pharmacological endpoint is enormously complicated. It is known that a number of drugs exert their pharmacological actions via drug-receptor interaction. The drug-receptor interaction is measurable, and the magnitude of the interaction depends on the affinity of the drug to the receptors as well as on the concentration of the drug in the biophase. The biophase concentration depends not only on the amount of drugs administered but on many pharmacokinetic factors including absorption, distribution, and elimination processes. The binding affinity of compounds to various receptors in the development of novel drugs has been evaluated mainly by in vitro radioligand binding in tissue membrane preparations and in intact cells. However, the in vitro receptor-binding characteristics may not necessarily assure pharmacological specificity in vivo because various pharmacokinetic and pharmacodynamic factors are not taken into account. In fact, Beauchamp et al. (1995) demonstrated that the affinities of 13 angiotensin II antagonists for angiotensin II subtype 1 receptor determined in vitro with rat adrenal membrane did not correlate well with in vivo pharmacological potency. Thus, despite the extensive use of in vitro assays for initial screening of antagonists, which may have potential therapeutic effects, the apparent in vitro affinities for a group of potent angiotensin II subtype-1 receptor antagonists may be not predictive of in vivo potencies. Therefore, the characterization of drug-receptor interaction under physiological conditions would provide more practical information for the evaluation of novel drugs.

Currently, new types of ₁ adrenoceptor antagonists that exhibit high selectivity to ₁ adrenoceptors in the prostate are receiving a great deal of attention in terms of developing effective therapeutic agents for bladder outlet obstruction with less vascular side effects in patients with benign prostatic hyperplasia. The ₁ adrenoceptor antagonist, tamsulosin, has been shown to antagonize potently ₁ adrenoceptor-mediated responses in the lower urinary tract and prostate (Honda et al., 1985; Honda and Nakagawa, 1986) and improve urinary obstruction in patients with benign prostatic hyperplasia with less incidence of orthostatic hypotension (Chapple, 1996; Wilde and McTavish, 1996). ₁ Adrenoceptors have been classified into several subtypes (Hieble et al., 1995; Michel et al., 1995). Previous in vitro receptor-binding studies have shown that tamsulosin is a more selective antagonist of _1A and _1D adrenoceptor subtypes than _1B subtype (Michel and Insel, 1994; Testa et al., 1995; Michel et al., 1996; Taguchi et al., 1997) and that [³H]tamsulosin may be a suitable radioligand to label ₁ adrenoceptors because it has a higher binding affinity and lower level of nonspecific binding than [³H]prazosin and [³H]bunazosin (Yazawa et al. 1992; Yamada et al., 1994b). However, the in vivo receptor-binding characteristics of this drug have been clarified minimally, and, to our knowledge, there have been no reports of an assay procedure for ₁ adrenoceptors under physiological conditions, except in a recent report by Yamada et al. (1998), who have shown briefly in vivo binding of [³H]tamsulosin in rat tissues. Therefore, the present study was performed to measure simultaneously ₁ adrenoceptors in various tissues of rats by [³H]tamsulosin in vivo and to examine whether this technique could be applied to characterize their receptor-binding specificities in relation to the pharmacokinetics of novel ₁ adrenoceptor antagonists to serve as therapeutically useful agents in bladder outlet obstruction as a result of benign prostatic hyperplasia.

	Experimental Procedures

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Materials. [³H]Tamsulosin ([³H]YM617, 2.08 TBq/mmol) was synthesized by Amersham Intl. (Buckinghamshire, England), and it was kindly provided by Drs. O. Inagaki and K. Honda (Yamanouchi Pharm. Co. Ltd., Ibaraki, Japan). [¹⁴C]Iodoantipyrine (2.0 GBq/mmol) was purchased from DuPont NEN (Wilmington, DE). The following drugs were kindly donated by the companies indicated: prazosin hydrochloride, Pfizer Pharm. Co. Ltd. (Tokyo, Japan) and tamsulosin hydrochloride, Yamanouchi Pharm. Co. Ltd. Phentolamine hydrochloride, yohimbine hydrochloride, and ()propranolol hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). All other drugs and materials were obtained from commercial sources.

Animals. Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan) weighing approximately 200 g were used. Rats were housed with a 12-h light/dark cycle and fed laboratory food and water ad libitum.

In Vivo [³H]Tamsulosin Binding. In vivo measurement of specific [³H]tamsulosin binding in rat tissues was performed as described for the in vivo measurement of calcium channel antagonist receptors in rat tissues (Uchida et al., 1995). Rats were anesthetized with diethyl ether, and [³H]tamsulosin at the dose of 1.3 nmol/kg (555 kBq in 150 µl of saline) was injected into the femoral vein. The animals were allowed to recover; they were then sacrificed by taking blood from the descending aorta under temporary anesthesia with diethyl ether 10 min after the injection to minimize the effect of metabolism of the radioligand. In the experiment examining the time course of [³H]tamsulosin binding, rats were sacrificed at 3, 10, 60, 120, and 240 min. A blood sample was taken from the descending aorta, and tissues (prostate, vas deferens, aorta, submaxillary gland, spleen, heart, lung, liver, kidney, and cerebral cortex) were rapidly removed. After dissection on ice, each tissue was homogenized in ice-cold 50 mM Tris-HCl buffer to a final tissue concentration of 10 mg/ml using a Kinematica Polytron homogenizer. Particulate-bound radioactivity was determined by rapid filtration of 1 to 3 ml of the homogenate over Whatman GF/C filters, which were washed subsequently with 2 ml of ice-cold buffer. The particulate-bound radioactivity was measured by a liquid scintillation counter after the addition of scintillation fluids (2 liters of toluene, 1 liter of Triton X-100, 15 g of 2,5-diphenyloxazole, and 0.3 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene). In this case, particulate-bound radioactivity of [³H]tamsulosin in each tissue from phentolamine (3.15, 31.5, 62.9, and 126 µmol/kg i.p., 0.5-h pretreatment)- or nifedipine (28.9 µmol/kg p.o., 1-h pretreatment)-administered rats was determined. Based on the data with pharmacological specificity, particulate-bound radioactivity from vehicle and phentolamine (62.9 µmol/kg i.p.)-pretreated rats was defined as total binding and nonspecific binding, respectively, and the difference could be determined as in vivo specific [³H]tamsulosin binding. In the preliminary experiment, it was shown that there was no significant difference in the amount of in vivo specific [³H]tamsulosin binding between once- and twice-washout with 2 ml of ice-cold buffer of Whatman GF/C filters after the filtration of tissue homogenates. Thus, we considered that nonspecifically bound radioactivity could be almost removed by once-washout with 2 ml of buffer under the present assay condition. The data were expressed as femtomole per milligram of tissue (wet weight).

In Vitro [³H]Tamsulosin Binding. The binding assay of [³H]tamsulosin in rat tissues was performed by a similar method as described previously in human prostates (Yamada et al., 1994b). The tissues (prostate, submaxillary gland, spleen, heart, lung, and kidney) were minced with scissors and homogenized by a Kinematica Polytron homogenizer in 20 to 80 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.5). The homogenates were centrifuged at 40,000g for 20 min. The pellet was resuspended in the ice-cold buffer, and the suspension was centrifuged again at 40,000g for 20 min. The resulting pellet was resuspended in the buffer for the binding assay. All steps were performed at 4°C. The tissue homogenates (5-10 mg of wet weight tissue) were incubated with [³H]tamsulosin (0.02-1.0 nM) in 50 mM Tris-HCl buffer (pH 7.5). Incubation was carried out for 30 min at 25°C. The reaction was terminated by rapid filtration (Cell Harvester; Brandel, Gaithersburg, MD) through Whatman GF/B glass fiber filters, and the filters were rinsed three times with 3 ml of ice-cold buffer. The tissue-bound radioactivity was extracted from the filters overnight in scintillation fluid (2 liters of toluene, 1 liter of Triton X-100, 15 g of 2,5-diphenyloxazole, and 0.3 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene), and the radioactivity was determined by a liquid scintillation counter. Specific binding of [³H]tamsulosin was determined experimentally from the difference between counts in the absence and presence of 10 µM phentolamine. All assays were conducted in duplicate. The analysis of binding data was performed as described previously (Yamada et al., 1980). K_d and B_max values for [³H]tamsulosin were estimated by Rosenthal analysis of the saturation data (Rosenthal, 1967).

Determination of [³H]Tamsulosin in Plasma. For determination of plasma levels, [³H]tamsulosin (555 kBq, 1.3 nmol/kg) was i.v. injected into the femoral vein of rats, and a small amount (100-800 µl) of blood was taken from the femoral artery through the cannula at 1, 3, 5, 10, 30, 60, and 120 min. The plasma was separated by centrifugation. Determination of [³H]tamsulosin concentration in plasma was performed by a modified version of the HPLC described previously by Soeishi et al. (1990). Four-hundred microliters of acetonitrile was added to plasma samples (50-400 µl) containing [¹⁴C]iodoantipyrine as an internal standard. After being stirred, the mixture was centrifuged at 8500g for 5 min. The supernatant was transferred to a test tube and evaporated to dryness under reduced pressure. The residue was dissolved in 150 µl of the mobile phase, and 50 µl of the solution was injected into the HPLC system. The HPLC system consisted of a pump (880-PU; Jasco, Tokyo, Japan), a stainless steel column (15 cm × 4.0 mm i.d.) packed with Nucleosil 5C₁₈ (Machery-Nagel, Düren, Germany). The mobile phase was 0.2 M potassium biphosphate, 0.2 M phosphoric acid, acetonitrile (7:7:5, v/v) at a flow rate of 1.0 ml/min. The column elute was collected in a vial, and radioactivity was measured by liquid scintillation counter.

Measurement of Blood Flow Rate with [¹⁴C]Iodoantipyrine. The blood flow rate in rat tissues was measured by a modification of the method described by Sakurada et al. (1978). Rats were anesthetized with diethylether, and the femoral vein and artery were catheterized. [¹⁴C]Iodoantipyrine was infused into the femoral vein at a constant rate (185 kBq/min) for 30 s. During this infusion period, eight arterial blood samples were periodically (at 4-s intervals) obtained from the arterial catheter, and blood radioactivity was measured. Rats were decapitated immediately after the 30-s infusion with [¹⁴C]iodoantipyrine, and prostate, aorta, spleen, heart, lung, liver, kidney, and cerebral cortex were dissected. Tissues were weighed, and the radioactivity was measured.

	Results

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Identification of In Vivo Specific Binding of [³H]Tamsulosin in Rat Tissues. The particulate-bound radioactivity was measured in rat tissues (prostate, vas deferens, aorta, cerebral cortex, submaxillary gland, spleen, heart, lung, liver, and kidney) 10 and 60 min after i.v. injection of [³H]tamsulosin (555 kBq, 1.3 nmol/kg). Pretreatment with phentolamine at doses of 3.15 and 31.5 µmol/kg (i.p.) reduced dose-dependently (31-74% and 71-94%, respectively) [³H]tamsulosin binding in particulate fractions of each tissue except the cerebral cortex and liver, which showed only a slight decrease; further significant decreases, compared with the reduction at 31.5 µmol/kg, were not seen by higher doses (62.9, 126 µmol/kg) of phentolamine (Fig. 1). As shown in Fig. 2, therefore, the difference in particulate-bound radioactivity of [³H]tamsulosin in each tissue between vehicle- and phentolamine (62.9 µmol/kg)-pretreated rats could be defined as in vivo specific binding of the ligand. The amount of nonspecific binding of [³H]tamsulosin, which was defined as binding in phentolamine-pretreated tissues, compared with specific binding, was much lower in all tissues except the cerebral cortex and liver. Ten and 60 min after i.v. injection of [³H]tamsulosin in rats, specific [³H]tamsulosin binding occurred in all tissues examined except the cerebral cortex and liver, which showed little significant amount of specific binding. The amount of specific [³H]tamsulosin binding differed markedly among tissues. The rank order of [³H]tamsulosin binding at 10 min after i.v. injection was kidney > lung, heart > submaxillary gland, spleen > aorta, vas deferens, and prostate. Administration (p.o.) of nifedipine (28.9 µmol/kg), a potent vasodilator, had little significant effect on the particulate-bound radioactivity in rat tissues after i.v. injection of [³H]tamsulosin (Fig. 2). The rank order of level of specific [³H]tamsulosin binding at 60 min after i.v. injection tended to be similar to that at 10 min, but the difference among tissues was smaller (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effects of pretreatment with varying doses of phentolamine on [3H]tamsulosin binding in rat tissues (, liver; , prostate; , aorta; , submaxillary gland; , spleen; , heart) 10 min after i.v. injection of the ligand. Rats received varying doses (3.15-126 µmol/kg i.p.) of phentolamine 30 min before i.v. injection of [3H]tamsulosin. [3H]Tamsulosin (555 kBq, 1.3 nmol/kg) was injected into the femoral vein, and rats were sacrificed at 10 min. [3H]Tamsulosin binding in particulate fraction of each tissue was determined, and it was expressed as percentage of control total binding of [3H]tamsulosin in each tissue from vehicle-pretreated rats. Each point represents mean ± S.D. of three rats.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effects of pretreatment with phentolamine and nifedipine on [3H]tamsulosin binding in rat tissues. Rats received vehicle (control), phentolamine (62.9 µmol/kg i.p.) at 30 min and nifedipine (28.9 µmol/kg p.o.) at 60 min before i.v. injection of [3H]tamsulosin. [3H]Tamsulosin (555 kBq, 1.3 nmol/kg) was injected into the femoral vein, and rats were sacrificed at 10 min; [3H]tamsulosin binding in particulate fractions of each tissue was then determined. Each column represents mean ± S.D. of three rats. Asterisks show a significant difference from control values; ***P < .001.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Time course of total concentration of [3H]tamsulosin in plasma after i.v. injection in rats. [3H]Tamsulosin (555 kBq, 1.3 nmol/kg) was injected into the femoral vein. Blood samples were taken from the femoral artery at 1 to 120 min. Each point represents mean ± S.D. of three rats.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time course of in vivo specific binding of [3H]tamsulosin in rat tissues (A: , submaxillary gland; , spleen; , aorta; , prostate; B: , kidney; , lung; , heart) after i.v. injection of the ligand. [3H]Tamsulosin (555 kBq, 1.3 nmol/kg) was injected into the femoral vein, and rats were sacrificed 3 to 240 min later. Specific binding of [3H]tamsulosin was experimentally defined as the difference in binding in particulate fractions of each tissue from vehicle- (total binding) and phentolamine- (62.9 µmol/kg, i.p.) pretreated (nonspecific binding) rats. Each point represents mean ± S.D. of three to six rats.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   In vivo total (), specific (), and nonspecific () binding of [3H]tamsulosin in the particulate fraction from rat prostate as a function of increasing free concentration of the ligand. A mixture of [3H]tamsulosin (555 kBq, 1.3 nmol/kg) and unlabeled tamsulosin at doses of 1.3 to 41.8 nmol/kg was injected into the femoral vein of vehicle- (total binding) and phentolamine- (62.9 µmol/kg i.p.) (nonspecific binding) pretreated rats, and the binding in particulate fraction of prostate was measured at 10 min. Each point represents mean ± S.D. of three rats. Solid lines show the computer-generated curves using the binding parameters listed in Table 1.

Competition Studies. A constant amount of [³H]tamsulosin (1.3 nmol/kg) was coinjected with increasing amounts of unlabeled tamsulosin, prazosin, terazosin, yohimbine, and propranolol in rats. Intravenous injection of low doses of tamsulosin (2.7-40.4 nmol/kg) and prazosin (7.2-71.6 nmol/kg) inhibited dose-dependently in vivo specific binding of [³H]tamsulosin in particulate fractions of the prostate, aorta, submaxillary gland, spleen, heart, lung, and kidney of rats. Figure 6 illustrates the dose-dependent inhibition curves by tamsulosin and prazosin in the prostate and spleen. Also, terazosin (6.5-652 nmol/kg) showed a significant inhibition of [³H]tamsulosin binding in each tissue (data not shown). The ID₅₀ values for tamsulosin and prazosin differed markedly not only among both drugs but among tissues (Table 2). Compared with those for prazosin, ID₅₀ values for tamsulosin were 12.4, 9.8, and 13.9 times smaller in the prostate, lung, and kidney, respectively, and 5 to 6 times smaller in the aorta, submaxillary gland, and heart. On the other hand, the ID₅₀ value for tamsulosin in the spleen was 1.6 times greater than that for prazosin. To examine tissue selectivity or ₁ subtype selectivity of tamsulosin and prazosin, we compared ratios of their ID₅₀ values in different rat tissues. The ratios of ID₅₀ (spleen) to ID₅₀ (submaxillary gland) of tamsulosin and prazosin were 1.02 and 0.11, respectively; the ratios of ID₅₀ (spleen) to ID₅₀ (prostate) were 1.33 and 0.07, respectively; and the ratios of ID₅₀ (aorta) to ID₅₀ (prostate) were 0.39 and 0.17, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   In vivo inhibition by tamsulosin () and prazosin () of specific [3H]tamsulosin binding in the prostate (A) and spleen (B) of rats. Tamsulosin (2.7-40.4 nmol/kg) and prazosin (7.2-71.6 nmol/kg) were injected into the femoral vein with [3H]tamsulosin (555 kBq, 1.3 nmol/kg), and rats were sacrificed at 10 min. Each point represents mean ± S.D. of three rats.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Effects of prazosin, yohimbine, and propranolol on specific [3H]tamsulosin binding in rat tissues. Prazosin (7.2, 71.6 nmol/kg), yohimbine (76.7, 256 nmol/kg), and propranolol (101, 338 nmol/kg) were injected into the femoral vein with [3H]tamsulosin (555 kBq, 1.3 nmol/kg), and rats were sacrificed at 10 min. Each column represents mean ± S.D. of three rats. Asterisks show a significant difference from control values: *P < .05; **P < .01; and ***P < .001.

In Vitro [³H]Tamsulosin Binding. K_d values for in vitro specific [³H]tamsulosin binding in homogenates of prostate, submaxillary gland, spleen, heart, lung, and kidney of rats were 52.7 ± 8.5, 59.8 ± 6.0, 170 ± 19, 107 ± 13, 59.7 ± 8.9, and 67.9 ± 7.9 pM, respectively, and B_max values were 1.37 ± 0.17, 6.08 ± 0.24, 2.78 ± 0.61, 3.52 ± 0.53, 3.50 ± 0.05, and 5.54 ± 0.76 fmol/mg tissue (mean ± S.D., n = 3), respectively. The K_d value in the spleen was 1.6 to 3.2 times greater than the K_d values in other tissues. B_max values were greatest in the submaxillary gland and kidney, followed by the heart, lung > spleen > prostate. As shown in Fig. 8, B_max values for [³H]tamsulosin in rat tissues from in vivo binding experiments (Table 1) correlated significantly with those from in vitro binding experiments. Although there was a similarity in K_d values for this ligand in the spleen, lung, and kidney between in vivo (Table 1) and in vitro, in vivo K_d (K_d(Cf)) values in the prostate and submaxillary gland were about 4 times greater than in vitro K_d values, and K_d (K_d(Cf)) in the heart was 2.8 times smaller.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Correlation between in vivo and in vitro in maximal number of binding sites (Bmax) for [3H]tamsulosin in rat tissues. In vivo Bmax values for [3H]tamsulosin in rat tissues (prostate, spleen, lung, heart, submaxillary gland, and kidney) were obtained from Table 1, and in vitro Bmax values were described in the text. In vivo Bmax values for [3H]tamsulosin are mean ± S.D. of 20 rats (Table 1), and those of in vitro Bmax are mean ± S.D. of three rats. The correlation coefficient (r) for this relationship was 0.90 (significant at P < .05).

Tissue Blood Flow. The local blood flow rates in the prostate, aorta, cerebral cortex, spleen, heart, lung, liver, and kidney of rats were 0.13 ± 0.03, 1.29 ± 0.54, 1.06 ± 0.04, 0.13 ± 0.09, 8.47 ± 4.17, 4.53 ± 1.12, 0.44 ± 0.25, and 1.92 ± 0.98 ml/min/g tissue (mean ± S.D., n = 3), respectively, thus varying markedly among tissues. The values in the prostate, spleen, heart, liver, and kidney of rats were reasonably close to previously reported blood flow levels in these tissues in rats (Gerlowski and Jain, 1983; Davies and Morris, 1993). The blood flow rate was highest in the heart, followed by the lung > kidney > aorta > cerebral cortex > liver > prostate and spleen.

	Discussion

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The present study was undertaken to simultaneously measure ₁ adrenoceptors in rat tissues by [³H]tamsulosin in vivo. The particulate-bound radioactivity was measured in homogenates of rat tissues 10 min after i.v. injection of [³H]tamsulosin. Pretreatment with phentolamine at the dose of 31.5 µmol/kg reduced markedly (71-94%) [³H]tamsulosin binding in particulate fractions of each tissue except the cerebral cortex and liver, and further decreases were not seen by two or four times higher doses of phentolamine. Thus, the difference in particulate-bound radioactivity of [³H]tamsulosin in each tissue between vehicle- and phentolamine (62.9 µmol/kg i.p.)-pretreated rats was defined as in vivo specific binding of the ligand. Although in vivo specific [³H]tamsulosin binding in rat tissues was unaffected by relatively high doses of yohimbine and propranolol, it was dose-dependently inhibited by the coinjection of low doses of unlabeled tamsulosin, prazosin, and terazosin with the radioligand. As a matter of fact, in vivo specific [³H]tamsulosin binding in the prostate and aorta was effectively inhibited by i.v. injections of tamsulosin (2.7-40.4 nmol/kg) and prazosin (7.2-71.6 nmol/kg). Recently, Martin et al. (1997) have evaluated the effects of several ₁ adrenoceptor antagonists on urethral and arterial pressures in conscious male rats under normal adrenergic tone, and they have shown significant decreases in both urethral and arterial pressures 5 to 15 min after i.v. injections of tamsulosin (24.6 and 74.0 nmol/kg) and prazosin (7.2 and 24.0 nmol/kg). In our preliminary experiment, tamsulosin at i.v. dose ranges that exhibited specific binding in the rat prostate inhibited dose-dependently the phenylephrine-induced increases in urethral pressure in anesthetized rats (S. Y. et al., unpublished observation). Based on the close correlation in i.v. dose ranges between in vivo ₁ adrenoceptor-binding activities of tamsulosin and prazosin in rat tissues and their functional activities, it seems likely that specific binding of [³H]tamsulosin in rat tissues after i.v. injection reflects in vivo selective labeling of the pharmacologically relevant ₁ adrenoceptors.

There is a possibility that i.p. administration of a high dose of phentolamine for the measurement of nonspecific binding of [³H]tamsulosin should cause vasodilation and thus affect the distribution of [³H]tamsulosin and other drugs given thereafter. Also, the low dose of tamsulosin, prazosin, and terazosin would have minimal effects on blood pressure in rats, but the high doses in saturation and competition studies could have cardiovascular effects that might alter the distribution of agents to various tissues. Thus, we examined the effect of nifedipine, a potent vasodilator, on in vivo [³H]tamsulosin binding. Pretreatment with nifedipine at p.o. dose of 28.9 µmol/kg, which caused a marked and sustained hypotension in rats (Yamanaka et al., 1991), had little significant effect on the particulate-bound radioactivity in each tissue of rats after i.v. injection of [³H]tamsulosin. Thus, it is unlikely that a hypotension due to the blockade of ₁ adrenoceptors has a significant effect on the tissue distribution of [³H]tamsulosin and other drugs given thereafter, and also on the subsequent ₁ adrenoceptor binding.

In vivo specific binding of [³H]tamsulosin 10 and 60 min after i.v. injection was widely distributed in various tissues, and the degree of nonspecific binding, compared with the specific binding, was markedly lower in tissues except the cerebral cortex and liver. Such low nonspecific binding of [³H]tamsulosin was in accord with in vitro binding data of this ligand obtained in rat and human tissues (Yazawa et al., 1992; Yamada et al., 1994b). Taken together, these data suggest that [³H]tamsulosin is a suitable ligand for in vivo labeling of ₁ adrenoceptors in rat tissues. Rat liver, unlike other tissues, exhibited a high level of nonspecific binding of [³H]tamsulosin. Soeishi et al. (1996) reported that tamsulosin after p.o. administration was rapidly metabolized in liver but hardly metabolized in other organs or plasma of rats. Thus, the reason why in vivo specific [³H]tamsulosin binding was little seen in the liver is possibly related to the extensive metabolism of the ligand in the tissue.

The binding parameters (K_d and B_max) for in vivo [³H]tamsulosin binding were estimated by injecting i.v. varying doses of unlabeled tamsulosin with [³H]tamsulosin into rats. B_max values for in vivo [³H]tamsulosin binding in the prostate, submaxillary gland, spleen, lung, heart, and kidney of rats were comparable with those from in vitro binding experiments (Fig. 8). Although there was a similarity between in vitro and in vivo in K_d values for [³H]tamsulosin in the spleen, lung, and kidney, in vivo K_d (K_d(Cf)) values in the prostate and submaxillary gland were about 4 times greater, and the value in the heart was 2.8 times smaller, than each in vitro K_d value. The reason for this discrepancy in these tissues is not clear at the present time. It is assumed that the Cf of tamsulosin for the estimation of in vivo K_d (K_d(Cf)) values might differ significantly from the extracellular concentration in the vicinity of receptors in these tissues. In this connection, it may be of a interest to note that among rat tissues examined, the blood flow rate was highest in the heart and lowest in the prostate. ₁ Adrenoceptors participate in the regulation of physiological responses in numerous tissues. Clinically, ₁ adrenoceptors are an important target for therapeutic manipulation as well as a possible site of pathologic etiology (Yamada et al., 1984, 1987). For these reasons, in vivo ₁ adrenoceptor-binding parameters for [³H]tamsulosin in rat tissues may be of potential use for characterizing not only pharmacological specificity of ₁ adrenoceptor antagonists but affinity and density of ₁ adrenoceptors under pathologic conditions such as cardiovascular diseases and lower urinary dysfunction.

A relatively high degree of in vivo specific binding of [³H]tamsulosin was observed in the heart, lung, and kidney of rats compared with other tissues including the prostate. Specific [³H]tamsulosin binding in the kidney, lung, heart, and spleen was greatest 3 min after i.v. injection, and it declined rapidly with the disappearance of [³H]tamsulosin from the plasma. On the other hand, in vivo [³H]tamsulosin binding in the prostate and aorta peaked at 60 and 10 min, respectively, with a considerable level of specific binding in both tissues persisting up to 240 min postinjection. The most sustained binding of [³H]tamsulosin occurred in the submaxillary gland. These data are consistent with our ex vivo observation that p.o. administration of tamsulosin in rats, despite a rapid decline in the plasma concentration, brought about more selective and sustained occupancy of ₁ adrenoceptors in the prostate and submaxillary gland than in the spleen and heart (Ohkura et al., 1998). Accordingly, the constant level of specific binding as a function of time for [³H]tamsulosin in the prostate and submaxillary gland of rats may be related to the relatively slow dissociation rate of this ligand from the receptor sites, possibly due to a high affinity for the receptors. However, it must be noted that pharmacokinetic factors such as blood flow rate and volume of distribution in each organ may be responsible for the observed difference among tissues in the amount and time course of in vivo specific [³H]tamsulosin binding. Tissue blood flow may be a critical determinant for in vivo binding of drugs to receptors. In fact, the level of blood flow rate measured by [¹⁴C]iodoantipyrine was greater in the heart, lung, and kidney than in other tissues.

It is known that _1A subtype exists predominantly in the submaxillary gland, vas deferens, and prostate of rats (Han et al., 1987; Michel et al., 1989; Testa et al., 1993; Yazawa and Honda, 1993; Lepor et al., 1994; Shibata et al., 1995; Ford et al., 1996), whereas _1B subtype is predominant in the spleen and liver (Han et al., 1987; Han and Minneman, 1991; Michel et al., 1993; Shibata et al., 1995). Based on estimated ID₅₀ values for in vivo [³H]tamsulosin binding, the inhibitory effect of tamsulosin in the prostate, lung, and kidney was 10 to 14 times greater than that of prazosin, and it was 5 to 6 times greater in the aorta, submaxillary gland, and heart. However, in the spleen, tamsulosin was 1.6 times less potent than prazosin. In the present experiment, it is likely that in vivo specific binding of [³H]tamsulosin in rat tissues exhibited already tissue selectivity because the drug binds to both _1A and _1D subtypes with higher affinity than to _1B subtype (Michel and Insel, 1994; Testa et al., 1995; Michel et al., 1996; Yamada et al., 1998; Ohkura et al. 1998). Prazosin is known generally as a nonselective antagonist of ₁ subtypes both in vitro and in vivo (Hanft and Gross, 1989; Aboud et al., 1993; Martin et al., 1997). To evaluate in vivo tissue selectivity or ₁ subtype selectivity of tamsulosin, therefore, it may be useful to compare the ratio of ID₅₀ value for the drug with that for prazosin among different tissues. Ratios of ID₅₀ (spleen) to ID₅₀ (submaxillary gland) of tamsulosin and prazosin were 1.02 and 0.11, respectively, and the ratios of ID₅₀ (spleen) to ID₅₀ (prostate) were 1.33 and 0.07, respectively. Thus, tamsulosin was 9 and 19 times, respectively, greater than prazosin in selectivity of ₁ adrenoceptors in the submaxillary gland and prostate versus the spleen. Consequently, these data may provide the first direct in vivo evidence that tamsulosin binds to _1A subtype with higher affinity than to _1B subtype.

Ratios of ID₅₀ (aorta) to ID₅₀ (prostate) of tamsulosin and prazosin in inhibiting specific [³H]tamsulosin binding were 0.39 and 0.17, respectively. The value of the ratio of ID₅₀ (aorta) to ID₅₀ (prostate) for tamsulosin divided by the ratio for prazosin was 2, and it may represent the relative selectivity of ₁ adrenoceptors in the prostate versus the aorta. In other words, tamsulosin may exhibit a 2-fold higher selectivity for ₁ adrenoceptors than does prazosin in the prostate as compared with the aorta. This difference was smaller than the in vitro difference (12 times) in ₁ adrenoceptor-binding affinity between the human prostate and aorta (Yamada et al., 1994a). Although there is no clear explanation for this difference at present, previous studies have shown that ₁ agonist-induced contractile responses of the rat aorta and human peripheral artery are mediated primarily via _1D and _1B subtypes, respectively (Aboud et al., 1993; Hatano et al., 1994; Kenny et al., 1995), and that tamsulosin exhibits considerably high affinity for _1D subtype (Testa et al., 1995; Michel et al., 1996; Noble et al., 1997; Taguchi et al., 1997). Accordingly, it is plausible that tamsulosin has a certain degree of affinity to ₁ adrenoceptors in the rat aorta. In conclusion, this study suggests that [³H]tamsulosin is a useful ligand not only for the characterization of ₁ adrenoceptors in various tissues under physiological conditions but for in vivo evaluation of novel ₁ adrenoceptor antagonists as potentially useful therapeutic agents in benign prostatic hyperplasia in terms of tissue selectivity and ₁ adrenoceptor subtype selectivity.