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A Pharmacological Study of Alpha Adrenergic Receptor Subtypes in Smooth Muscle of Human Urinary Bladder Base and Prostatic Urethra
0022-534 7/85/1342-0396$02.00/0 Vol. 134, August
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright © 1985 by The Williams & Wilkins Co.
A PHARMACOLOGICAL STUDY OF ALPHA ADRENERGIC RECEPTOR SUBTYPES IN SMOOTH MUSCLE OF HUMAN URINARY BLADDER BASE AND PROSTATIC URETHRA YOSHITAKA KUNISAWA,* KAZUKI KAWABE, TADAO NIIJIMA, KAZUO HONDA TOICHI TAKENAKA
AND
From the Department of Urology, University of Tokyo and the Department of Pharmacology, Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan
ABSTRACT
Postsynaptic alpha adrenergic receptor subtypes mediating contraction of human urinary bladder base and prostatic urethra were investigated in vitro. Alpha adrenergic receptor agonists, noradrenaline and phenylephrine, induced contraction dose-dependently in these tissues. Alpha adrenergic receptor antagonists, prazosin and yohimbine, competitively antagonized the contraction induced by these agonists in the bladder base as well as prostatic urethra. The mean pA 2 values for the antagonists in the bladder base and prostatic urethra were as follows: 8.89 and 8.96 for prazosin and 6.30 and 6.45 for yohimbine, respectively. Comparison of pA 2 values of prazosin and yohimbine in both the tissues with those values found in the literature for these compounds in a variety of tissues containing alpha-1 or alpha-2 adrenergic receptor indicates that the postsynaptic alpha adrenergic receptor subtype in the human urinary bladder base and prostatic urethra is the alpha1 type, not the alpha-2 type. It is well known that the nerve supplies to the bladder are mainly parasympathetic, and partly sympathetic. Recent histochemical and pharmacological studies suggest that the urinary bladder neck and urethra have the same innervation. Ahlquist1 classified adrenergic receptors into alpha and beta adrenergic receptors. There have since been many reports classifying the bladder and urethra as an alpha or beta adrenergic receptor. It is generally accepted that alpha adrenergic receptors predominate in the proximal urethra and bladder base, 2- 5 while beta adrenergic receptors are the predominant adrenergic receptor type in the urinary bladder body. Lands et al. 6 classified beta adrenergic receptors into beta-1, which is chiefly distributed in the heart, and beta-2, which is chiefly distributed in vessels and bronchi. Langer 7 found that alpha adrenergic receptors existed not only in postsynaptic but also in presynaptic sites which inhibited releasing chemical transmitter noradrenaline. Postsynaptic alpha adrenergic receptors were designated as alpha-1 adrenergic receptors and presynaptic alpha adrenergic receptors were designated as alpha-2 adrenergic receptors. Subsequently, it has been recommended that the classification alpha-1 and alpha-2 should not be used independently of the location and function of alpha adrenergic receptors, but rather according to the relative affinity for agonists and antagonists. There are many reports concerning the participation of either alpha-1 or alpha-2 adrenoceptor in the various human tissues, but no reports are available for the smooth muscle of the human urinary bladder base and prostatic urethra. The purpose of the present study is to determine the subtype of the alpha adrenergic receptor in those human tissues. MATERIALS AND METHODS
Materials. Human urinary bladder base and prostatic urethra were obtained when total cystectomies were done in ten male patients whose ages ranged from 68 to 79, the average being 74 years, and in one female who was 74 years old. The dissected portions of urinary bladder base and prostatic urethra were Accepted for publication March 25, 1985. * Requests for reprints: Dept. of Urology, Branch Hospital, University of Tokyo, Mejidoro 3-28-6, Bunkyo-ku, Tokyo 112, Japan.
immediately put in 4C Krebs-Henseleit solution and kept in the solution for a period of about 12 hours before in vitro experiments were started. Muscle strip preparation. The muscle strips from the urinary bladder base and prostatic urethra were cut to equal length (about 7 X 3 mm.) transversely and gently dissected free of mucosa, adipose tissues and the prostatic gland. Twenty-six muscle strips of bladder base were obtained from four male patients and one female patient. Thirty-two muscle strips of prostatic urethra were obtained from nine patients. The composition of Krebs-Henseleit solution was (in mM): NaCl, 118.4; KCl, 4.7; CaCb, 2.5; KH2P04, 1.2; MgS04, 1.2; NaHC0 3 , 25, and glucose, 11.1. In all cases, Krebs-Henseleit solution contained 4 X 10-5 M corticosterone and 10-s M desmethylimipramine which inhibited extraneuronal and neuronal uptake of noradrenaline, respectively, and 10-5 M propranolol to block beta adrenergic receptors. Each muscle strip was suspended in a single chamber containing Krebs-Henseleit solution with corticosterone, desmethylimipramine and propranolol at 37C, continuously aerated with a 95 per cent 0 2 -5 per cent CO 2 mixture, and allowed to equilibrate under appropriate resting tension (1 gm.) for at least 1 hr. before drug addition. The contraction of the muscle strips with various pharmacologic agents was recorded simultaneously through an isometric force displacement transducer (FD pickup, Nihon-Koden) on an inkwriting oscillograph (T02N2, Fujisoku). Drugs. All drugs were prepared immediately before the experiments in distilled and demineralized water or saline; prazosin (PR) hydrochloride (Pfizer), yohimbine (YO) hydrochloride (Sigma), (-)-noradrenaline (NA) hydrochloride and (-)phenylephrine (PE) hydrochloride (Tokyo Kasei). Dose response curve. We confirmed that one tissue strip was capable of three dose-response curves. We gave each strip a one hour-interval and thorough wash of agonists between doses. For example, the maximum contractions for NA were 1.6 gm. (100 per cent), 1.5 gm. (94 per cent), 1.5 gm. (94 per cent) and 1.4 gm. (88 per cent), and for PE the maximum contractions were 1.7 gm. (100 per cent), 1.5 gm. (91 per cent) and 1.4 gm. (85 per cent). Then these strips could reproduce the doseresponse curves for NA and PE. Dose response curves for a-agonists, NA and PE were determined by increasing concen396
397
ALPHA ADRENERGiC RECEPTOR SUBTYPES IN BLADDER BASE AND PROSTAT!C URETHRA
trations of the agonist approximately 3-fold in the baths. Before the cumulative dose-response curves were obtained, submaximal contractions were first elicited by repeated concentrations of agonists until constant responses were reached. After completion of a dose-response curve, the agonists were washed 3 times and left in the bath for an hour. The antagonists were added to the baths after a dose-response curve for the agonist had been obtained. The tissues were exposed to the antagonist for 30 min. before rechallenge with the agonists. Determination of dissociation constants. Dissociation constants (KB) of antagonists, PR and YO were determined according to the technique of Arunlakshana and Schild,8 by using the agonists, NA and PE. The dose ratios (ED 50 of the agonist in the presence of the antagonist divided by the control agonist ED 50 ) were determined. The equation relating the dissociation constant of a competitive antagonist to the dose ratio and the antagonist concentration (M) is: KB = Antagonist [M]/(Dose ratio - 1)
The KB values for PR and YO were determined each time, and the pA2 values (-log KB) were calculated. Schild plot. According to Arunlakshana and Schild,8 if blockade is competitive, a plot of the logarithm of (dose ratio - 1) against the negative logarithm of molar concentration of the antagonist should yield a straight line whose slope is unity. We examined the slope in all muscle strips by the regression line method. Statistical evaluation. The results were expressed as the mean ± S.D. Statistical differences between the slope and unity were tested by using Student's t test under null hypothesis (slope= 1).
iOO
50
~
" u
-Log Noradrenaline (M)
.£
1! "E 0
100
100
50
50
()
J -Log Phenylephrine (M)
FIG. 2. Dose response curves for prazosin and yohimbine against noradrenaline and ,phenylephrine _in_ prost!ltic urethra under 4 x 10-5 corticosterone, 10- M desmethyhm1pramme and 10-5 M propranolol (see METHODS). Results are mean± S.D. of 3 to 16 experiments.
TABLE 1.
Prostatic Urethra
100
100
50
50
"
0
No.
Noradrenaline
No.
Phenylephrine
8.88 ± 0.22 1.03 (0.808-1.25) 6.21 ± 0.21 0.958 (0.591-1.32)
11
8.90 ± 0.21 0.789 (0.510-1.07) 6.41 ± 0.23 0.759 (0.427-1.09)
Prazosin
pA," slopeb
16
Yohimbine
pA, slope
10
12
Urinary Bladder Base Agonist
Antagonist No.
Noradrenaline
No.
Phenylephrine
8.86 ± 0.28 0.927 (0.593-1.26) 6.36 ± 0.13 0.929 (0.714-1.14)
10
8.63 ± 0.22 0.731' (0.467-0.994) 6.11 ± 0.28 0.910 (0.442-1.38)
Prazosin
pA, slope
13
Yohimbine
pA, slope
12
12
"pA, values are means ± S.D. b Slopes of Schild plot are means with 95% confidence limits. All slopes without care not significantly different from unity (significant level= 0.05). No. = The number of experiments.
Dose response curves to PR and YO in the urinary bladder base and the prostatic urethra are shown in figures 1 and 2. pA2 values for PR and YO against NA and PE in the urinary bladder base and prostatic urethra of humans are given in table 1. Schild plots for PR and YO as competitive antagonists of NA and PE in the bladder base and prostatic urethra are demonstrated in table l and figure 3.
-Log Noradrenaline (M)
~
DISCUSSION
~
"EO
Agonist
Antagonist
RESULTS
NA (1 x 10- 7 to 3 X 10- 4 M) had a contractile effect on the urinary bladder base and the prostatic urethra. Their maximum contractions were 2.06 ± 0.82 gm. (no. = 11) and 2.33 ± 0. 77 gm. (no. = 16), respectively. The tissue response to the drug was dose related and the mean ED 50 ± S.D. for the urinary bladder base and the prostatic urethra was (1.42 ± 0.37) X 10-6 M (no.= 11) and (2.09 ± 0.76) x 10-6 M (no.= 16), respectively. PE (3 x 10-6 to 3 x 10-3 M) also had a contractile effect on the urinary bladder base and the prostatic urethra. Their maximum contractions were 2.85 ± 0.83 gm. (no. = 15) and 2.52 ± 0.91 gm. (no. = 16), respectively. The tissue response to the drug was dose-related and the mean ED5o ± S.D. for the urinary bladder base and the prostatic urethra was (4.16 ± 1.91) x 10-6 M (no.= 15) and (3.70 ± 1.91) x 10-6 M (no.= 16), respectively.
pA 2 values for prazosin and yohimbine in the human prostatic urethra and urinary bladder base
100
100
50
50
()
Yoh;mb;a~w0
1
3
10 x10 6 M
1 -Log Phenylephrine (M)
FIG. 1. Dose response curves for prazosin and yohimbine against noradrenaline and phenylephrine in urinary bladder base under 4 X 10-5 M corticosterone, 10-s M desmethylimipramine and 10-5 M propranolol (see METHODS). Results are mean ± S.D. of 3 to 16 experiments.
It is evident that alpha receptors exist in the urinary bladder base and in the prostatic urethra. This is why alpha"blockers are used as remedies for patients with difficulty in voiding. However, alpha blockers can be classified into non-selective alpha blockers (such as phentolamine), selective alpha-1 blockers (such as PR), and selective alpha-2 blockers (such as YO). One way to find out which of the above three groups is the most effective for improving voiding difficulty is to identify the subtype of alpha receptors in the tissue. For this purpose we tried to identify the subtype of the alpha receptors in specimens of human smooth muscle of the urinary bladder base and prostatic urethra.
398
KUNISAWA AND ASSOCIATES Human Prostatic Urethra
2
Agonist= Phenylephrine
Agonisl= Noradrenaline
Prazosin Prazosm
Yohimbine
hboe-
,___ _.,___ _
10
~-
7
6
-~ 5
Human Urinary Bladder Base
:rgon1st=Phenylephrlne
Agonist= Noradrenaline
Yohimbine
Prazosin
Yohimbine
Prazostn
10
In the clinical data, Thien et al. 11 reported that urinary incontinence was caused by PR. On the other hand Takita et al. 12 reported the effect of prazosin on the difficulty due to prostatic hypertrophy: there was a statistically significant reduction in residual urine and in bladder compliance, and the maximum cystometric capacity increased in the majority of patients after the treatment. Anderson et al. 13 and Okumura et al. 14 reported decline of the urethral pressure in the urethral pressure profile with the use of PR. Thus these clinical data also suggested that there were alpha-1 receptors in the urinary bladder base and prostatic urethra and that the selective alpha1 antagonist was effective for patients with prostatic hypertrophy and bladder neck contraction. We conclude that contraction in the smooth muscles of the human urinary bladder base and prostatic urethra is mediated by alpha-1 adrenergic receptors. Acknowledgments. The authors gratefully acknowledge the skillful technical assistance of Miss Akemi Osawa. REFERENCES
-Log Antagonist (M)
FIG. 3. Schild plots for prazosin and yohimbine in urinary bladder base and prostatic urethra. The results are mean ± S.D. of 3 to 6 experiments.
First we investigated the dose-response relationship to NA and PE in the urinary bladder base and prostatic urethra. Marked contractions were noted in both tissues. These contractions could be blocked by PR, a selective alpha-1 receptor antagonist, and YO, a selective alpha-2 receptor antagonist and relatively weak postsynaptic alpha-1 antagonist (fig. 1, fig. 2). One of the most reliable techniques to differentiate receptors and receptor subtypes is the comparison of dissociation constants of agonists or antagonists. Therefore, we determined the pA 2 of PR and YO (table 1). Doxey et al. 9 reported that the pA 2 of PR is equal to 8.2 in the case of an alpha-1 blocker, while the pA 2 is less than 6.62 in the case of an alpha-2 blocker, and that the pA2 of YO is 6.4 for alpha-1 receptors and is 8.4 for alpha-2 receptors. Ruffolo et al. 10 studied the pA 2 of YO in the literature, and revealed that the pA2 of YO was 5.52 to 6. 70 in alpha-1 receptors and 7.33 to 8.3 in alpha-2 receptors. The results of the present study revealed that the pA2 of PR was between 8.63 and 8.90, while that of YO was between 6.11 and 6.41. These figures and a review of the literature demonstrate that the smooth muscles of the urinary bladder base and prostatic urethra seem to contract with alpha-1 but not alpha2 receptors. Plots of the logarithm of (dose ratio - 1) against the negative logarithm of the molar concentration of the antagonist are shown in figure 3. If blockade is competitive, they yield a straight line whose slope is unity. When we used NA and PE as agonists in both tissues, most of the slopes of the Schild plot for PR and YO were not significantly different from unity (significant level = 0.05) indicating that blockade by prazosin and yohimbine was competitive in nature. All data suggest that there are only pure alpha-1 types participating in the contraction of the smooth muscle of urinary bladder base and prostatic urethra.
1. Ahlquist, R. P.: The study of the adrenergic receptors. Am. J. Physiol., 153: 586, 1948. 2. Edvardsen, P. and Seteklein, J.: Distribution of adrenergic receptors in the urinary bladder of cats, rabbits and guinea-pigs. Acta Pharmacol. Toxicol., 26: 437, 1968. 3. Salmini, M., Seteklein, J. and Skobba, T. J.: The sensitivity of adrenergic excitatory and inhibitory receptors in the smooth muscle of the rabbit urinary bladder. Acta Pharmacol. Toxicol., 27: 213, 1969. 4. Downie, J. W., Dean, D. M., Carro-Ciampi, G. and Awad, S. A.: A difference in sensitivity to alpha adrenergic agonists exhibited by detrusor and bladder neck of rabbit. Can. J. Physiol. Pharmacol., 53: 525, 1975. 5. Levin, R. M. and Wein, A. J.: Distribution and function of adrenergic receptors in the urinary bladder of the rabbit. Mol. Pharmacol., 16: 441, 1979. 6. Lands, A. M., Luduena, F. P. and Buzzo, H.J.: Differentiation of receptors responsible to isoproterenol. Life Sci., 6: 2241, 1967. 7. Langer, S. Z.: Presynaptic receptors and their role in the regulation of transmitter release. Br. J. Pharmacol., 60: 481, 1977. 8. Arunlakshana, 0. and Schild, H. 0.: Some quantitative uses of drug antagonists. Br. J. Pharmacol., 14: 48, 1959. 9. Doxey, J. C., Smith, C. F. C. and Walker, J. M.: Selectivity of blocking agents for pre and postsynaptic alpha adrenoceptors. Br. J. Pharmacol., 60: 91, 1977. 10. Ruffolo, R. R. Jr., Waddell, J.E. and Yaden, E. L.: Postsynaptic alpha adrenergic receptor subtypes differentiated by yohimbine in tissue from the rat. Existence of alpha-2 adrenergic receptors in rat aorta. J. Pharmacol. Exp. Ther., 217: 235, 1981. 11. Thien, T. H., Delaere, K. P. J., Debruyne, F. M. and Koene, R. A. P.: Urinary incontinence caused by prazosin. Br. Med. J., 11: 622, 1978. 12. Takita, T., Otani, T., Kondo, A. and Mitsuya, H.: Urodynamic study of lower urinary tract. XII. The effect of prazosin hydrochloride in the treatment of prostatic obstruction. Jpn. J. Urol., 7 4: 1, 1983. (Abstract in English) 13. Anderson, K. E., Ek, A., Hedlund, H. and Mattiasson, A.: Effects ofprazosin on isolated human urethra and in patients with lower motor neuron lesion. Invest. Urol., 19: 39, 1981. 14. Okumura, K., Takamatsu, T. and Koyanagi, T.: Management of chronic spinal cord injury patients with prazosin. Jpn. J. Urol., 7 4: 1621, 1983. (Abstract in English)
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright © 1985 by The Williams & Wilkins Co.
A PHARMACOLOGICAL STUDY OF ALPHA ADRENERGIC RECEPTOR SUBTYPES IN SMOOTH MUSCLE OF HUMAN URINARY BLADDER BASE AND PROSTATIC URETHRA YOSHITAKA KUNISAWA,* KAZUKI KAWABE, TADAO NIIJIMA, KAZUO HONDA TOICHI TAKENAKA
AND
From the Department of Urology, University of Tokyo and the Department of Pharmacology, Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan
ABSTRACT
Postsynaptic alpha adrenergic receptor subtypes mediating contraction of human urinary bladder base and prostatic urethra were investigated in vitro. Alpha adrenergic receptor agonists, noradrenaline and phenylephrine, induced contraction dose-dependently in these tissues. Alpha adrenergic receptor antagonists, prazosin and yohimbine, competitively antagonized the contraction induced by these agonists in the bladder base as well as prostatic urethra. The mean pA 2 values for the antagonists in the bladder base and prostatic urethra were as follows: 8.89 and 8.96 for prazosin and 6.30 and 6.45 for yohimbine, respectively. Comparison of pA 2 values of prazosin and yohimbine in both the tissues with those values found in the literature for these compounds in a variety of tissues containing alpha-1 or alpha-2 adrenergic receptor indicates that the postsynaptic alpha adrenergic receptor subtype in the human urinary bladder base and prostatic urethra is the alpha1 type, not the alpha-2 type. It is well known that the nerve supplies to the bladder are mainly parasympathetic, and partly sympathetic. Recent histochemical and pharmacological studies suggest that the urinary bladder neck and urethra have the same innervation. Ahlquist1 classified adrenergic receptors into alpha and beta adrenergic receptors. There have since been many reports classifying the bladder and urethra as an alpha or beta adrenergic receptor. It is generally accepted that alpha adrenergic receptors predominate in the proximal urethra and bladder base, 2- 5 while beta adrenergic receptors are the predominant adrenergic receptor type in the urinary bladder body. Lands et al. 6 classified beta adrenergic receptors into beta-1, which is chiefly distributed in the heart, and beta-2, which is chiefly distributed in vessels and bronchi. Langer 7 found that alpha adrenergic receptors existed not only in postsynaptic but also in presynaptic sites which inhibited releasing chemical transmitter noradrenaline. Postsynaptic alpha adrenergic receptors were designated as alpha-1 adrenergic receptors and presynaptic alpha adrenergic receptors were designated as alpha-2 adrenergic receptors. Subsequently, it has been recommended that the classification alpha-1 and alpha-2 should not be used independently of the location and function of alpha adrenergic receptors, but rather according to the relative affinity for agonists and antagonists. There are many reports concerning the participation of either alpha-1 or alpha-2 adrenoceptor in the various human tissues, but no reports are available for the smooth muscle of the human urinary bladder base and prostatic urethra. The purpose of the present study is to determine the subtype of the alpha adrenergic receptor in those human tissues. MATERIALS AND METHODS
Materials. Human urinary bladder base and prostatic urethra were obtained when total cystectomies were done in ten male patients whose ages ranged from 68 to 79, the average being 74 years, and in one female who was 74 years old. The dissected portions of urinary bladder base and prostatic urethra were Accepted for publication March 25, 1985. * Requests for reprints: Dept. of Urology, Branch Hospital, University of Tokyo, Mejidoro 3-28-6, Bunkyo-ku, Tokyo 112, Japan.
immediately put in 4C Krebs-Henseleit solution and kept in the solution for a period of about 12 hours before in vitro experiments were started. Muscle strip preparation. The muscle strips from the urinary bladder base and prostatic urethra were cut to equal length (about 7 X 3 mm.) transversely and gently dissected free of mucosa, adipose tissues and the prostatic gland. Twenty-six muscle strips of bladder base were obtained from four male patients and one female patient. Thirty-two muscle strips of prostatic urethra were obtained from nine patients. The composition of Krebs-Henseleit solution was (in mM): NaCl, 118.4; KCl, 4.7; CaCb, 2.5; KH2P04, 1.2; MgS04, 1.2; NaHC0 3 , 25, and glucose, 11.1. In all cases, Krebs-Henseleit solution contained 4 X 10-5 M corticosterone and 10-s M desmethylimipramine which inhibited extraneuronal and neuronal uptake of noradrenaline, respectively, and 10-5 M propranolol to block beta adrenergic receptors. Each muscle strip was suspended in a single chamber containing Krebs-Henseleit solution with corticosterone, desmethylimipramine and propranolol at 37C, continuously aerated with a 95 per cent 0 2 -5 per cent CO 2 mixture, and allowed to equilibrate under appropriate resting tension (1 gm.) for at least 1 hr. before drug addition. The contraction of the muscle strips with various pharmacologic agents was recorded simultaneously through an isometric force displacement transducer (FD pickup, Nihon-Koden) on an inkwriting oscillograph (T02N2, Fujisoku). Drugs. All drugs were prepared immediately before the experiments in distilled and demineralized water or saline; prazosin (PR) hydrochloride (Pfizer), yohimbine (YO) hydrochloride (Sigma), (-)-noradrenaline (NA) hydrochloride and (-)phenylephrine (PE) hydrochloride (Tokyo Kasei). Dose response curve. We confirmed that one tissue strip was capable of three dose-response curves. We gave each strip a one hour-interval and thorough wash of agonists between doses. For example, the maximum contractions for NA were 1.6 gm. (100 per cent), 1.5 gm. (94 per cent), 1.5 gm. (94 per cent) and 1.4 gm. (88 per cent), and for PE the maximum contractions were 1.7 gm. (100 per cent), 1.5 gm. (91 per cent) and 1.4 gm. (85 per cent). Then these strips could reproduce the doseresponse curves for NA and PE. Dose response curves for a-agonists, NA and PE were determined by increasing concen396
397
ALPHA ADRENERGiC RECEPTOR SUBTYPES IN BLADDER BASE AND PROSTAT!C URETHRA
trations of the agonist approximately 3-fold in the baths. Before the cumulative dose-response curves were obtained, submaximal contractions were first elicited by repeated concentrations of agonists until constant responses were reached. After completion of a dose-response curve, the agonists were washed 3 times and left in the bath for an hour. The antagonists were added to the baths after a dose-response curve for the agonist had been obtained. The tissues were exposed to the antagonist for 30 min. before rechallenge with the agonists. Determination of dissociation constants. Dissociation constants (KB) of antagonists, PR and YO were determined according to the technique of Arunlakshana and Schild,8 by using the agonists, NA and PE. The dose ratios (ED 50 of the agonist in the presence of the antagonist divided by the control agonist ED 50 ) were determined. The equation relating the dissociation constant of a competitive antagonist to the dose ratio and the antagonist concentration (M) is: KB = Antagonist [M]/(Dose ratio - 1)
The KB values for PR and YO were determined each time, and the pA2 values (-log KB) were calculated. Schild plot. According to Arunlakshana and Schild,8 if blockade is competitive, a plot of the logarithm of (dose ratio - 1) against the negative logarithm of molar concentration of the antagonist should yield a straight line whose slope is unity. We examined the slope in all muscle strips by the regression line method. Statistical evaluation. The results were expressed as the mean ± S.D. Statistical differences between the slope and unity were tested by using Student's t test under null hypothesis (slope= 1).
iOO
50
~
" u
-Log Noradrenaline (M)
.£
1! "E 0
100
100
50
50
()
J -Log Phenylephrine (M)
FIG. 2. Dose response curves for prazosin and yohimbine against noradrenaline and ,phenylephrine _in_ prost!ltic urethra under 4 x 10-5 corticosterone, 10- M desmethyhm1pramme and 10-5 M propranolol (see METHODS). Results are mean± S.D. of 3 to 16 experiments.
TABLE 1.
Prostatic Urethra
100
100
50
50
"
0
No.
Noradrenaline
No.
Phenylephrine
8.88 ± 0.22 1.03 (0.808-1.25) 6.21 ± 0.21 0.958 (0.591-1.32)
11
8.90 ± 0.21 0.789 (0.510-1.07) 6.41 ± 0.23 0.759 (0.427-1.09)
Prazosin
pA," slopeb
16
Yohimbine
pA, slope
10
12
Urinary Bladder Base Agonist
Antagonist No.
Noradrenaline
No.
Phenylephrine
8.86 ± 0.28 0.927 (0.593-1.26) 6.36 ± 0.13 0.929 (0.714-1.14)
10
8.63 ± 0.22 0.731' (0.467-0.994) 6.11 ± 0.28 0.910 (0.442-1.38)
Prazosin
pA, slope
13
Yohimbine
pA, slope
12
12
"pA, values are means ± S.D. b Slopes of Schild plot are means with 95% confidence limits. All slopes without care not significantly different from unity (significant level= 0.05). No. = The number of experiments.
Dose response curves to PR and YO in the urinary bladder base and the prostatic urethra are shown in figures 1 and 2. pA2 values for PR and YO against NA and PE in the urinary bladder base and prostatic urethra of humans are given in table 1. Schild plots for PR and YO as competitive antagonists of NA and PE in the bladder base and prostatic urethra are demonstrated in table l and figure 3.
-Log Noradrenaline (M)
~
DISCUSSION
~
"EO
Agonist
Antagonist
RESULTS
NA (1 x 10- 7 to 3 X 10- 4 M) had a contractile effect on the urinary bladder base and the prostatic urethra. Their maximum contractions were 2.06 ± 0.82 gm. (no. = 11) and 2.33 ± 0. 77 gm. (no. = 16), respectively. The tissue response to the drug was dose related and the mean ED 50 ± S.D. for the urinary bladder base and the prostatic urethra was (1.42 ± 0.37) X 10-6 M (no.= 11) and (2.09 ± 0.76) x 10-6 M (no.= 16), respectively. PE (3 x 10-6 to 3 x 10-3 M) also had a contractile effect on the urinary bladder base and the prostatic urethra. Their maximum contractions were 2.85 ± 0.83 gm. (no. = 15) and 2.52 ± 0.91 gm. (no. = 16), respectively. The tissue response to the drug was dose-related and the mean ED5o ± S.D. for the urinary bladder base and the prostatic urethra was (4.16 ± 1.91) x 10-6 M (no.= 15) and (3.70 ± 1.91) x 10-6 M (no.= 16), respectively.
pA 2 values for prazosin and yohimbine in the human prostatic urethra and urinary bladder base
100
100
50
50
()
Yoh;mb;a~w0
1
3
10 x10 6 M
1 -Log Phenylephrine (M)
FIG. 1. Dose response curves for prazosin and yohimbine against noradrenaline and phenylephrine in urinary bladder base under 4 X 10-5 M corticosterone, 10-s M desmethylimipramine and 10-5 M propranolol (see METHODS). Results are mean ± S.D. of 3 to 16 experiments.
It is evident that alpha receptors exist in the urinary bladder base and in the prostatic urethra. This is why alpha"blockers are used as remedies for patients with difficulty in voiding. However, alpha blockers can be classified into non-selective alpha blockers (such as phentolamine), selective alpha-1 blockers (such as PR), and selective alpha-2 blockers (such as YO). One way to find out which of the above three groups is the most effective for improving voiding difficulty is to identify the subtype of alpha receptors in the tissue. For this purpose we tried to identify the subtype of the alpha receptors in specimens of human smooth muscle of the urinary bladder base and prostatic urethra.
398
KUNISAWA AND ASSOCIATES Human Prostatic Urethra
2
Agonist= Phenylephrine
Agonisl= Noradrenaline
Prazosin Prazosm
Yohimbine
hboe-
,___ _.,___ _
10
~-
7
6
-~ 5
Human Urinary Bladder Base
:rgon1st=Phenylephrlne
Agonist= Noradrenaline
Yohimbine
Prazosin
Yohimbine
Prazostn
10
In the clinical data, Thien et al. 11 reported that urinary incontinence was caused by PR. On the other hand Takita et al. 12 reported the effect of prazosin on the difficulty due to prostatic hypertrophy: there was a statistically significant reduction in residual urine and in bladder compliance, and the maximum cystometric capacity increased in the majority of patients after the treatment. Anderson et al. 13 and Okumura et al. 14 reported decline of the urethral pressure in the urethral pressure profile with the use of PR. Thus these clinical data also suggested that there were alpha-1 receptors in the urinary bladder base and prostatic urethra and that the selective alpha1 antagonist was effective for patients with prostatic hypertrophy and bladder neck contraction. We conclude that contraction in the smooth muscles of the human urinary bladder base and prostatic urethra is mediated by alpha-1 adrenergic receptors. Acknowledgments. The authors gratefully acknowledge the skillful technical assistance of Miss Akemi Osawa. REFERENCES
-Log Antagonist (M)
FIG. 3. Schild plots for prazosin and yohimbine in urinary bladder base and prostatic urethra. The results are mean ± S.D. of 3 to 6 experiments.
First we investigated the dose-response relationship to NA and PE in the urinary bladder base and prostatic urethra. Marked contractions were noted in both tissues. These contractions could be blocked by PR, a selective alpha-1 receptor antagonist, and YO, a selective alpha-2 receptor antagonist and relatively weak postsynaptic alpha-1 antagonist (fig. 1, fig. 2). One of the most reliable techniques to differentiate receptors and receptor subtypes is the comparison of dissociation constants of agonists or antagonists. Therefore, we determined the pA 2 of PR and YO (table 1). Doxey et al. 9 reported that the pA 2 of PR is equal to 8.2 in the case of an alpha-1 blocker, while the pA 2 is less than 6.62 in the case of an alpha-2 blocker, and that the pA2 of YO is 6.4 for alpha-1 receptors and is 8.4 for alpha-2 receptors. Ruffolo et al. 10 studied the pA 2 of YO in the literature, and revealed that the pA2 of YO was 5.52 to 6. 70 in alpha-1 receptors and 7.33 to 8.3 in alpha-2 receptors. The results of the present study revealed that the pA2 of PR was between 8.63 and 8.90, while that of YO was between 6.11 and 6.41. These figures and a review of the literature demonstrate that the smooth muscles of the urinary bladder base and prostatic urethra seem to contract with alpha-1 but not alpha2 receptors. Plots of the logarithm of (dose ratio - 1) against the negative logarithm of the molar concentration of the antagonist are shown in figure 3. If blockade is competitive, they yield a straight line whose slope is unity. When we used NA and PE as agonists in both tissues, most of the slopes of the Schild plot for PR and YO were not significantly different from unity (significant level = 0.05) indicating that blockade by prazosin and yohimbine was competitive in nature. All data suggest that there are only pure alpha-1 types participating in the contraction of the smooth muscle of urinary bladder base and prostatic urethra.
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