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Response of the In Vitro Whole Bladder (Rabbit) Preparation to Autonomic Agonists
c-022 .. 5347;s2;~285-l087$02.oo;o 128, No·vernber
THE JOURNAL OF UROLOGY
Copyright© 1982 by The Willia.n1s & VVilkins Co.
Print€d in U.S. A.
RESPONSE OF THE IN VITRO WHOLE BLADDER PREPARATION TO AUTONOMIC AGONISTS ROBERT M. LEVIN* AND ALAN J. WEIN From the Division of Urology, University of Pennsylvania School of Medicine and the Philadelphia Veterans Administration M~edical Center, Philadelphia, Pennsylvania
ABSTRACT
Using an in vitro whole bladder preparation (rabbit), we studied both the volume-pressure relationship and the response of the bladder to specific autonomic agonists. We determined the response in: 1) an isovolumetric system in which the intravesical pressure was monitored in the absence of volume changes; 2) a system in which volume alterations were monitored in the presence of increasing pressure; and 3) a system in which the bladder emptied in the presence of a constant pressure. The results of these studies demonstrated: 1) volume-pressure studies generated a typical cystometric curve; 2) bethanechol produced a dose-dependent increase in int.ravesical pressure (decrease in bladder volume); 3) the whole bladder responded very poorly to isoproterenol; and 4) methoxamine and ATP both produced a modest increase in intravesical pressure. One advantage of the "whole bladder" in vitro preparation is that it can objectively measure a functional response of the bladder to pharmacological agents. Much of our knowledge of the response of the urinary bladder to pharmacological agents comes from in vitro muscle bath studies. 1- 3 These studies measure the isometric response of bladder smooth muscle strips to specific pharmacological agents. These types of studies have been very useful in determining the length-tension relationship of bladder smooth muscle,4 response to electrical stimulation 5 and pharmacological response to a variety of agonists and antagonists. 1- 3• e- 7 One potential source of error in the interpretation of studies using muscle strip methodology is related to the orientation of the muscle. In vivo, the bladder contraction consists of both isometric and isotonic properties: the initial contraction when the outlet is closed could be considered isometric (isovolumic) whereas the expulsion of the urine would be more isotonic since it involves the actual shortening of fibers against a specific pressure. In addition, the length-tension data obtained for muscle strips may not be directly applicable to the presswre-volume relationship of the intact bladder. The following studies were designed to measure accurately the pressure-volume relationship in an in vitro whole bladder preparation and determine the response of this preparation to autonomic agonists. M:ATERJ:ALS AND METHODS
Whole
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Male New Zealand WJ:,ite rabbits (2 were anesthetized with ketami.ne-xylazine (25 mg. ketamine, 6 mg The urinary bladders were exposed through a midline uR,,.,,,vu, the ureters tied, and the bladder was excised as I.ow on the urethra as possible. The bladder was emptied and the urethra canulated with a saline-filled 5-mm. tygon tube. The bladder was mounted in a 300-ml. isolated bath chamber containing Tyrode solution (NaCl [125 mM], KCl mM], NaH2P04 [0.4 mM], CaCb [1.8 mM], MgCl2 [0.5 mM], and NaHC03 [23.8 mM]) equilibrated with 95 per cent 02, 5 per cent CO2 at 37C (see fig. 1).
The bladder was connected to a Statham pressure transducer via a 3-way stopcock. As shown in figure 1, the 3rd connection to the pressure transducer's 3-way valve is to a saline reservoir housed in a 100-ml. graduated bmette. The bmette is mounted on a stand so that it can be raised or lowered quickly and easily. The pressure transducer and bladder base are always maintained at the same level. With the transducer valve open (to both bladder and burette) the level of saline in the burette is set at the same level as the transducer and bladder base; at this point, the pressure is set at zero (volume= 0). After a 30-minute equilibrium period, the burette is raised in 10-ml. (6-cm.) increments. After each increment, the bladder is allowed to equilibrate as the saline enters (5 minutes). At equilibrium both the volume which entered the bladder and the pressure is recorded. Through this methodology, we determined the volume-pressure relationship (cystometry) for the isolated bladder, which is, of course, independent of all neuronal influences. This can be determined for the "open" system in which the transducer is continually open to both bladder and burette or for the "closed" system in which the transducer is connected only to the bladder for pressure detenninations, i.e., the valve is open between burette and bladder for and then switched to bladder and transducer for pressure determination. For the pharmacological studies, 1 of 3 systems was used: "Closed . The bladder is filled to a volume via raising burette as previously descxibed" tion at the volume, the valve is rotated so that the transducer is connected to bladder (the system is "closed" to the burette). The is then added to the bath in a cumulative manner at 5-minute intervals. In this system the volume of the bladder cannot change and thus we are measuring isometric (isovolumic) contraction. "Open system": The bladder is filled to a specific volume via raising the burette as previously described. After equilibration at the specific volume, the cl.rugs are added to the bath. In this system the bladder is allowed to contract and expel the saline back into the burette; thus, the bladder volume changes simultaneously with pressure changes (since the pressure within the system is equal to difference in height between the pressure transducer [and bladder] and the height of fluid in the burette at equilibrium, the change in saline level equals the change in pressure). "Expulsion system": The bladder is filled to a specific volume
Accepted for publication June 9, 1982 Supported in part by grant RO-AM-2-6508-01 from the National Institutes of Health, grants from the Veterans Administration, and the McCabe Fund. * Requests for reprints: Division of Urology, 3010 Ravdin Courtyard Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104. 1087
LEVIN AND WEIN
1088 Expulsion Tube
;
100ml Graduated Surette {Reservoir)
6.5cm
,I
Fm. 1. Diagram of whole-bladder apparatus. Schematic diagram of apparatus used for in vitro whole bladder preparation.
via raising the burette as previously described. After equilibration the burette valve is closed and the saline-filled expulsion tube is connected to the system as pictured in figure 1. The top of the expulsion tube is set equal to the saline level in the burette, thus maintaining the same pressure in the system. With the bladder open to the expulsion tube, the drugs are added to the bath as previously described. In this system the bladder is allowed to contract and expel urine from the system without altering the saline column height. Thus the pressure in the static system is constant and the ability of the bladder to expel urine against a specific static pressure can be determined. The bladder expels saline into a beaker on a pan balance; the weight of the saline expelled is then converted into the volume expelled.
Isolated Muscle Strip Preparation The urinary bladders of 8 male New Zealand White rabbits were removed under ketamine-xylazine anesthesia. The bladder was rapidly dissected free of fat, and the body and base were separated at the level of the ureteral orifices. The bladder body was divided into 4 longitudinal strips of equal size which were placed in individual muscle baths containing 30 ml. of Tyrode's solution containing glucose (1 mg./ml.) and equilibrated with a gas mixture of 95 per cent 02 and 5 per cent CO2. After 20 minutes of equilibration, 1 gm. of tension was placed on each strip, and the strips were maintained in the oxygenated buffer for an additional 10 minutes. At the end of the equilibration period, dose-response curves to bethanechol and isoproterenol were generated via the cumulative addition of drug to the muscle bath at 5 minute intervals.
Initially, the bladder is filled with either 10, 25 or 50 ml. of saline. For the closed system (fig. 3) the bladder is disconnected from the burette after the filling is completed; thus the pressure response to bethanechol reflects the isovolumic contraction of the rabbit bladder. In the open system, bethanechol stimulates the movement of the saline into the burette (fig. 4). The increase in pressure reflects the height to which the column of saline within the burette rises. In the expulsion system, the volume expelled from the bladder is presented in figure 5. Although there were temporal changes in pressure during expulsion, the static pressure (pressure between drug additions) remained constant. In all cases, bethanechol produced a dose-dependent increase in bladder contraction. The largest responses in the closed and open systems were produced in the bladders filled with 10 ml. of saline, whereas the bladders containing 50 ml. responded the poorest. In this regard, it is interesting to note that the greatest pressure that the bladder was able to generate was approximately 14 mm.Hg. If the total pressure obtained for each curve is calculated, they range between 10 and 14 mm.Hg. In the expulsion system, the bladder was able to expel 90+ per cent of the saline at all 3 initial volumes, although the higher volumes required greater concentrations ofbethanechol.
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40
50
60
Volume (ml.)
Fm. 2. Volume-pressure curve (open system). The relationship between intravesical volume and pressure for the isolated rabbit bladder. Intravesical pressure was determined at specific bladder volumes. This curve represents the average of 8 separate experiments (8 different preparations).
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Initial Volume
(Initial Pressure}
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25mls
(5.8mm Hg)
50mls
(10.5mm Hg)
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RESULTS
Volume-pressure curves. Figure 2 displays the volume-pressure profile curve (n = 8) (open system). This appears to be a typical cystometric filling curve. The initial pressure rises (from zero) rapidly with the 1st addition of volume. Additional increases in volume (up to 45 ml.) produce little change in pressure. Above 45 ml., the pressure rises rapidly up to approximately 18 mm.Hg. At a critical pressure (between 16 and 20 mm.Hg) the bladder becomes overstretched, at which point the pressure drops rapidly and the volume increases (data not shown). Volume-pressure curves for the closed system are virtually identical with that presented for the open system (data not shown). Response to bethanechol. The response of the whole bladder preparation to bethanechol is presented in Figures 3, 4 and 5.
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10
100
1000
Bethanechol (µM)
Fm. 3. Effect of bethanechol (closed system). The dose reponse of the bladder to bethanechol was determined for the "closed" system in which the intravesical pressure was determined in the absence of volume changes. Each point represents the mean ± SE for 5 separate preparations.
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potency of bethanechol in th£ ~r-p~,Q,UH gTeater than that of either the open or
I
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of the whole bladder to isoproterenol, methoxamine and ATP (closed system). The bladder responded very
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to the addition of isoproterenol and moderately to methoxamine and ATP. Maximal response to these agents is presented in figure 6. (n = 4-6)
-5
DISCUSSION
., a::
In-vitro studies of the response of smooth muscle strips obtained from the rabbit and canine urinary bladder to specific autonomic agonists demonstrated that the response was dependent upon where the bladder strip was obtained. 6- 7 Smooth muscle strips obtained from the bladder dome responded substantially greater to bethanechol (muscarinic-cholinergic agonist), isoproterenol (beta-adrenergic agonist) and ATP (purinergic agonist) than strips obtained from the mid-bladder or bladder base, whereas the bladder base responded substantially greater to methoxamine (alpha-adrenergic) than did the midbladder or bladder body. One problem in relating this data to what happens in the whole bladder is related to the orientation of the muscle strips. Muscle strip experiments measure the 1dimensional isometric tension response to drugs, whereas the actual bladder response consists of 3-dimensional alterations in both tension and length which are reflected by changes in both intravesical pressure and volume. Early studies by Carpenter8- 10 used an in vitro whole bladder preparation (rat) in which pressure changes were used to quantitate the response of the bladder to both pharmacological
100 Initial Vo!ume
~
(\nitiot--llP Final Pressure)
10ml
(4.5mm Hg
25ml
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50ml
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-P
10mm Hg)
40
20
0
0
100
10 Bethanechol
1000
(pM)
Fm. 4. Effect of bethanechol (open system). The dose response of the bladder to bethanechol was determined for the "open" system as stated in Materials and Methods. Each point represents the mean ± SE for 5 separate preparations. 100
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0 0
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100
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BETHANECHOL
FIG. 5. Effect ofbethanechol (expulsion system). The dose response of the bladder to bethanechol was determined for the "expulsion" system as stated in Materials and Methods. Each point represents the mean ± SE for 5 separate preparations.
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Me!hoxamine
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Belhanechol
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(lmM)
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FIG. 6. Response to isoproterenol, methoxamine, ATP and bethanechoL The maximum response of the whole bladder preparation to isoproterenol, methoxamine, ATP and bethanecl:oL Each point represents the mean ± SE for 4 to 6 separate preparat10ns.
agents and electrical stimulation. Although these studies were extremely innovative and produced significant new information on the response of the bladder to a variety of agents, the whole bladder model was not adopted other researchers and has not been used subsequently. Based on these studies Carpenter, the present study describes the in vitro response of a whole bladder preparation to specific autonomic agonists. The rabbit was chosen for these studies because of the convenient size and volume of the bladder. Additionally, the rabbit bladder is rather thin and is maintained very 'Nell in the oxygenated bath. Unlike the studies Carpenter, which only monitored intravesical pressure, our setup as described can measure: 1) isovolumic (isometric) changes in intravesical pressure; 2) druginduced volume changes with accompanying pressure changes; and 3) isotonic changes in volume. Using our in vitro wholebladder preparation, we can determine the volume-pressure responses of the bladder to specific autonomic agonists. Thus, the volume-pressure relationship can be studied in the absence of neuronal reflexes present in the in vivo system. Our initial volrune,-pre1,strre studies on the whole bladder generated a standard ""'"11.-,u curve,8 1) an initial. rapid rise in pressure concomitant with the start of bladder filling; 2) a large plateau with a increase in nrP<: accompanying a rather increase volume; and a rise in pressure with increases in volume. At a specific pressme between 14 20 mm.Hg, the bladder would become overstretched and unstable, and result in a rapid fall in pressure and a rise in volume. Bethanechol produced a dose-related contraction of the bladder. In the closed (isovolumic), open, and expulsion systems the maximal pressure that the bladder could generate was approximately 14 mm.Hg (approximately the same maximal pressure obtained in our initial volume-pressure studies). The apparent differential response for the 10, 25 and 50 ml.-filled bladders in the open and closed systems can be explained as follows. The maximal pressure the bladder could generate was 14 mm.Hg. Thus, the 10 ml.-filled bladders that had the lowest initial pressure could respond to a greater degree than the 25 ml-filled bladder, which had an intermediate initial pressure. In the same manner, the 25 ml-filled bladders responded to a greater degree than the bladders containing 50 ml., which had the highest initial pressure.
1090
LEVIN AND WEIN
In the expulsion system, although the 10, 25 and 50 ml-filled bladders were all capable of full expulsion of the saline, there was an apparent difference in potency. This difference in potency can be explained as follows. The 10 and 25 ml.-filled bladder can be fully emptied by concentrations of bethanechol that are substantially below the concentrations that produce maximum contraction. The 50 ml.-filled bladder required a greater contractile force to empty and thus required a greater concentration of bethanechol. Graphing the data as the percentage of total volume expelled shows an apparent shift in potency between the 10 ml. (and 25 ml.) and 50 ml.-filled bladders. In conclusion, we feel that the in vitro whole bladder preparation has significant advantages when compared to the muscle strip preparations when relating the in vitro responses of the urinary bladder to the in-vivo effects of drugs on the bladder. Additionally, the expulsion system should be extremely useful in studying the effects of specific pathologies directly on in vitro bladder function.
2. 3.
4. 5. 6. 7. 8.
Acknowledgment. We would like to thank Ms. Janice High and Ms. Michelle Goldman for their excellent technical assistance. REFERENCES 1. Raezer, D. M., Wein, A. J., Jacobowitz, D. and Corriere, J. N., Jr.:
9. 10.
Autonomic innervation of canine urinary bladder. Urology, 2: 211, 1973. Edvardsen, P. and Seteklein, J.: Distribution of adrenergic receptors in the urinary bladder of cats, rabbits, and guinea pigs. Acta Pharmacol. Toxicol., 16: 437, 1968. 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. Hellstrand, P. and Johansson, B.: Analysis of the length response to a force step in smooth muscle from rabbit urinary bladder. Acta Physiol. Scand., 106: 221, 1979. Dean, D. M. and Downie, J. W.: Contribution of adrenergic and purinergic neurotransmission to contraction in rabbit detrusor. J. Pharmacol. Exp. Ther., 207: 431, 1978. 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. Levin, R. M., Shofer, F. S. and Wein, A. J.: Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmacol. Exp. Ther., 212: 536, 1980. Carpenter, F. G.: Excitation of rat urinary bladder by coaxial electrodes and by chemical agents. Am. J. Physiol., 204: 727, 1963. Carpenter, F. G. and Rand, S. A.: Relation of acetylcholine release to responses of the rat urinary bladder. J. Physiol., 180: 371, 1965. Carpenter, F. G. and Rubin, R. M.: The motor innervation of the rat urinary bladder. J. Physiol., 192: 609, 1967.
THE JOURNAL OF UROLOGY
Copyright© 1982 by The Willia.n1s & VVilkins Co.
Print€d in U.S. A.
RESPONSE OF THE IN VITRO WHOLE BLADDER PREPARATION TO AUTONOMIC AGONISTS ROBERT M. LEVIN* AND ALAN J. WEIN From the Division of Urology, University of Pennsylvania School of Medicine and the Philadelphia Veterans Administration M~edical Center, Philadelphia, Pennsylvania
ABSTRACT
Using an in vitro whole bladder preparation (rabbit), we studied both the volume-pressure relationship and the response of the bladder to specific autonomic agonists. We determined the response in: 1) an isovolumetric system in which the intravesical pressure was monitored in the absence of volume changes; 2) a system in which volume alterations were monitored in the presence of increasing pressure; and 3) a system in which the bladder emptied in the presence of a constant pressure. The results of these studies demonstrated: 1) volume-pressure studies generated a typical cystometric curve; 2) bethanechol produced a dose-dependent increase in int.ravesical pressure (decrease in bladder volume); 3) the whole bladder responded very poorly to isoproterenol; and 4) methoxamine and ATP both produced a modest increase in intravesical pressure. One advantage of the "whole bladder" in vitro preparation is that it can objectively measure a functional response of the bladder to pharmacological agents. Much of our knowledge of the response of the urinary bladder to pharmacological agents comes from in vitro muscle bath studies. 1- 3 These studies measure the isometric response of bladder smooth muscle strips to specific pharmacological agents. These types of studies have been very useful in determining the length-tension relationship of bladder smooth muscle,4 response to electrical stimulation 5 and pharmacological response to a variety of agonists and antagonists. 1- 3• e- 7 One potential source of error in the interpretation of studies using muscle strip methodology is related to the orientation of the muscle. In vivo, the bladder contraction consists of both isometric and isotonic properties: the initial contraction when the outlet is closed could be considered isometric (isovolumic) whereas the expulsion of the urine would be more isotonic since it involves the actual shortening of fibers against a specific pressure. In addition, the length-tension data obtained for muscle strips may not be directly applicable to the presswre-volume relationship of the intact bladder. The following studies were designed to measure accurately the pressure-volume relationship in an in vitro whole bladder preparation and determine the response of this preparation to autonomic agonists. M:ATERJ:ALS AND METHODS
Whole
UUUJ,!Ucf
,u,e:rn'J.flf,/,f,,fm.
Male New Zealand WJ:,ite rabbits (2 were anesthetized with ketami.ne-xylazine (25 mg. ketamine, 6 mg The urinary bladders were exposed through a midline uR,,.,,,vu, the ureters tied, and the bladder was excised as I.ow on the urethra as possible. The bladder was emptied and the urethra canulated with a saline-filled 5-mm. tygon tube. The bladder was mounted in a 300-ml. isolated bath chamber containing Tyrode solution (NaCl [125 mM], KCl mM], NaH2P04 [0.4 mM], CaCb [1.8 mM], MgCl2 [0.5 mM], and NaHC03 [23.8 mM]) equilibrated with 95 per cent 02, 5 per cent CO2 at 37C (see fig. 1).
The bladder was connected to a Statham pressure transducer via a 3-way stopcock. As shown in figure 1, the 3rd connection to the pressure transducer's 3-way valve is to a saline reservoir housed in a 100-ml. graduated bmette. The bmette is mounted on a stand so that it can be raised or lowered quickly and easily. The pressure transducer and bladder base are always maintained at the same level. With the transducer valve open (to both bladder and burette) the level of saline in the burette is set at the same level as the transducer and bladder base; at this point, the pressure is set at zero (volume= 0). After a 30-minute equilibrium period, the burette is raised in 10-ml. (6-cm.) increments. After each increment, the bladder is allowed to equilibrate as the saline enters (5 minutes). At equilibrium both the volume which entered the bladder and the pressure is recorded. Through this methodology, we determined the volume-pressure relationship (cystometry) for the isolated bladder, which is, of course, independent of all neuronal influences. This can be determined for the "open" system in which the transducer is continually open to both bladder and burette or for the "closed" system in which the transducer is connected only to the bladder for pressure detenninations, i.e., the valve is open between burette and bladder for and then switched to bladder and transducer for pressure determination. For the pharmacological studies, 1 of 3 systems was used: "Closed . The bladder is filled to a volume via raising burette as previously descxibed" tion at the volume, the valve is rotated so that the transducer is connected to bladder (the system is "closed" to the burette). The is then added to the bath in a cumulative manner at 5-minute intervals. In this system the volume of the bladder cannot change and thus we are measuring isometric (isovolumic) contraction. "Open system": The bladder is filled to a specific volume via raising the burette as previously described. After equilibration at the specific volume, the cl.rugs are added to the bath. In this system the bladder is allowed to contract and expel the saline back into the burette; thus, the bladder volume changes simultaneously with pressure changes (since the pressure within the system is equal to difference in height between the pressure transducer [and bladder] and the height of fluid in the burette at equilibrium, the change in saline level equals the change in pressure). "Expulsion system": The bladder is filled to a specific volume
Accepted for publication June 9, 1982 Supported in part by grant RO-AM-2-6508-01 from the National Institutes of Health, grants from the Veterans Administration, and the McCabe Fund. * Requests for reprints: Division of Urology, 3010 Ravdin Courtyard Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104. 1087
LEVIN AND WEIN
1088 Expulsion Tube
;
100ml Graduated Surette {Reservoir)
6.5cm
,I
Fm. 1. Diagram of whole-bladder apparatus. Schematic diagram of apparatus used for in vitro whole bladder preparation.
via raising the burette as previously described. After equilibration the burette valve is closed and the saline-filled expulsion tube is connected to the system as pictured in figure 1. The top of the expulsion tube is set equal to the saline level in the burette, thus maintaining the same pressure in the system. With the bladder open to the expulsion tube, the drugs are added to the bath as previously described. In this system the bladder is allowed to contract and expel urine from the system without altering the saline column height. Thus the pressure in the static system is constant and the ability of the bladder to expel urine against a specific static pressure can be determined. The bladder expels saline into a beaker on a pan balance; the weight of the saline expelled is then converted into the volume expelled.
Isolated Muscle Strip Preparation The urinary bladders of 8 male New Zealand White rabbits were removed under ketamine-xylazine anesthesia. The bladder was rapidly dissected free of fat, and the body and base were separated at the level of the ureteral orifices. The bladder body was divided into 4 longitudinal strips of equal size which were placed in individual muscle baths containing 30 ml. of Tyrode's solution containing glucose (1 mg./ml.) and equilibrated with a gas mixture of 95 per cent 02 and 5 per cent CO2. After 20 minutes of equilibration, 1 gm. of tension was placed on each strip, and the strips were maintained in the oxygenated buffer for an additional 10 minutes. At the end of the equilibration period, dose-response curves to bethanechol and isoproterenol were generated via the cumulative addition of drug to the muscle bath at 5 minute intervals.
Initially, the bladder is filled with either 10, 25 or 50 ml. of saline. For the closed system (fig. 3) the bladder is disconnected from the burette after the filling is completed; thus the pressure response to bethanechol reflects the isovolumic contraction of the rabbit bladder. In the open system, bethanechol stimulates the movement of the saline into the burette (fig. 4). The increase in pressure reflects the height to which the column of saline within the burette rises. In the expulsion system, the volume expelled from the bladder is presented in figure 5. Although there were temporal changes in pressure during expulsion, the static pressure (pressure between drug additions) remained constant. In all cases, bethanechol produced a dose-dependent increase in bladder contraction. The largest responses in the closed and open systems were produced in the bladders filled with 10 ml. of saline, whereas the bladders containing 50 ml. responded the poorest. In this regard, it is interesting to note that the greatest pressure that the bladder was able to generate was approximately 14 mm.Hg. If the total pressure obtained for each curve is calculated, they range between 10 and 14 mm.Hg. In the expulsion system, the bladder was able to expel 90+ per cent of the saline at all 3 initial volumes, although the higher volumes required greater concentrations ofbethanechol.
20
1 E
16
.§.
;
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12
ti
8
"'!!! g .s
4
ct
.!/
0
10
0
(n
= 5).
30
40
50
60
Volume (ml.)
Fm. 2. Volume-pressure curve (open system). The relationship between intravesical volume and pressure for the isolated rabbit bladder. Intravesical pressure was determined at specific bladder volumes. This curve represents the average of 8 separate experiments (8 different preparations).
~
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I ~
g
Initial Volume
(Initial Pressure}
10 mis
( 4.0 mm Hg)
25mls
(5.8mm Hg)
50mls
(10.5mm Hg)
10
RESULTS
Volume-pressure curves. Figure 2 displays the volume-pressure profile curve (n = 8) (open system). This appears to be a typical cystometric filling curve. The initial pressure rises (from zero) rapidly with the 1st addition of volume. Additional increases in volume (up to 45 ml.) produce little change in pressure. Above 45 ml., the pressure rises rapidly up to approximately 18 mm.Hg. At a critical pressure (between 16 and 20 mm.Hg) the bladder becomes overstretched, at which point the pressure drops rapidly and the volume increases (data not shown). Volume-pressure curves for the closed system are virtually identical with that presented for the open system (data not shown). Response to bethanechol. The response of the whole bladder preparation to bethanechol is presented in Figures 3, 4 and 5.
20
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0
0
10
100
1000
Bethanechol (µM)
Fm. 3. Effect of bethanechol (closed system). The dose reponse of the bladder to bethanechol was determined for the "closed" system in which the intravesical pressure was determined in the absence of volume changes. Each point represents the mean ± SE for 5 separate preparations.
1NaS '"c;"'H'--a"''-"Y
potency of bethanechol in th£ ~r-p~,Q,UH gTeater than that of either the open or
I
system.
of the whole bladder to isoproterenol, methoxamine and ATP (closed system). The bladder responded very
0, :i::
to the addition of isoproterenol and moderately to methoxamine and ATP. Maximal response to these agents is presented in figure 6. (n = 4-6)
-5
DISCUSSION
., a::
In-vitro studies of the response of smooth muscle strips obtained from the rabbit and canine urinary bladder to specific autonomic agonists demonstrated that the response was dependent upon where the bladder strip was obtained. 6- 7 Smooth muscle strips obtained from the bladder dome responded substantially greater to bethanechol (muscarinic-cholinergic agonist), isoproterenol (beta-adrenergic agonist) and ATP (purinergic agonist) than strips obtained from the mid-bladder or bladder base, whereas the bladder base responded substantially greater to methoxamine (alpha-adrenergic) than did the midbladder or bladder body. One problem in relating this data to what happens in the whole bladder is related to the orientation of the muscle strips. Muscle strip experiments measure the 1dimensional isometric tension response to drugs, whereas the actual bladder response consists of 3-dimensional alterations in both tension and length which are reflected by changes in both intravesical pressure and volume. Early studies by Carpenter8- 10 used an in vitro whole bladder preparation (rat) in which pressure changes were used to quantitate the response of the bladder to both pharmacological
100 Initial Vo!ume
~
(\nitiot--llP Final Pressure)
10ml
(4.5mm Hg
25ml
(5.6mm Hg--e>l3mm Hg)
50ml
(10mm Hg-f>' 14rnm Hg)
-P
10mm Hg)
40
20
0
0
100
10 Bethanechol
1000
(pM)
Fm. 4. Effect of bethanechol (open system). The dose response of the bladder to bethanechol was determined for the "open" system as stated in Materials and Methods. Each point represents the mean ± SE for 5 separate preparations. 100
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1000
BETHANECHOL
FIG. 5. Effect ofbethanechol (expulsion system). The dose response of the bladder to bethanechol was determined for the "expulsion" system as stated in Materials and Methods. Each point represents the mean ± SE for 5 separate preparations.
12
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10
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4
2
0 lsoprnierenol
Me!hoxamine
ATP
Belhanechol
(100.AJMl
(lmM)
(lmM)
(I00;.1M)
FIG. 6. Response to isoproterenol, methoxamine, ATP and bethanechoL The maximum response of the whole bladder preparation to isoproterenol, methoxamine, ATP and bethanecl:oL Each point represents the mean ± SE for 4 to 6 separate preparat10ns.
agents and electrical stimulation. Although these studies were extremely innovative and produced significant new information on the response of the bladder to a variety of agents, the whole bladder model was not adopted other researchers and has not been used subsequently. Based on these studies Carpenter, the present study describes the in vitro response of a whole bladder preparation to specific autonomic agonists. The rabbit was chosen for these studies because of the convenient size and volume of the bladder. Additionally, the rabbit bladder is rather thin and is maintained very 'Nell in the oxygenated bath. Unlike the studies Carpenter, which only monitored intravesical pressure, our setup as described can measure: 1) isovolumic (isometric) changes in intravesical pressure; 2) druginduced volume changes with accompanying pressure changes; and 3) isotonic changes in volume. Using our in vitro wholebladder preparation, we can determine the volume-pressure responses of the bladder to specific autonomic agonists. Thus, the volume-pressure relationship can be studied in the absence of neuronal reflexes present in the in vivo system. Our initial volrune,-pre1,strre studies on the whole bladder generated a standard ""'"11.-,u curve,8 1) an initial. rapid rise in pressure concomitant with the start of bladder filling; 2) a large plateau with a increase in nrP<:
1090
LEVIN AND WEIN
In the expulsion system, although the 10, 25 and 50 ml-filled bladders were all capable of full expulsion of the saline, there was an apparent difference in potency. This difference in potency can be explained as follows. The 10 and 25 ml.-filled bladder can be fully emptied by concentrations of bethanechol that are substantially below the concentrations that produce maximum contraction. The 50 ml.-filled bladder required a greater contractile force to empty and thus required a greater concentration of bethanechol. Graphing the data as the percentage of total volume expelled shows an apparent shift in potency between the 10 ml. (and 25 ml.) and 50 ml.-filled bladders. In conclusion, we feel that the in vitro whole bladder preparation has significant advantages when compared to the muscle strip preparations when relating the in vitro responses of the urinary bladder to the in-vivo effects of drugs on the bladder. Additionally, the expulsion system should be extremely useful in studying the effects of specific pathologies directly on in vitro bladder function.
2. 3.
4. 5. 6. 7. 8.
Acknowledgment. We would like to thank Ms. Janice High and Ms. Michelle Goldman for their excellent technical assistance. REFERENCES 1. Raezer, D. M., Wein, A. J., Jacobowitz, D. and Corriere, J. N., Jr.:
9. 10.
Autonomic innervation of canine urinary bladder. Urology, 2: 211, 1973. Edvardsen, P. and Seteklein, J.: Distribution of adrenergic receptors in the urinary bladder of cats, rabbits, and guinea pigs. Acta Pharmacol. Toxicol., 16: 437, 1968. 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. Hellstrand, P. and Johansson, B.: Analysis of the length response to a force step in smooth muscle from rabbit urinary bladder. Acta Physiol. Scand., 106: 221, 1979. Dean, D. M. and Downie, J. W.: Contribution of adrenergic and purinergic neurotransmission to contraction in rabbit detrusor. J. Pharmacol. Exp. Ther., 207: 431, 1978. 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. Levin, R. M., Shofer, F. S. and Wein, A. J.: Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmacol. Exp. Ther., 212: 536, 1980. Carpenter, F. G.: Excitation of rat urinary bladder by coaxial electrodes and by chemical agents. Am. J. Physiol., 204: 727, 1963. Carpenter, F. G. and Rand, S. A.: Relation of acetylcholine release to responses of the rat urinary bladder. J. Physiol., 180: 371, 1965. Carpenter, F. G. and Rubin, R. M.: The motor innervation of the rat urinary bladder. J. Physiol., 192: 609, 1967.