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Effect of Anoxia on Urethral Response to Phenylephrine
~22-5347/95/1544-1527$03.00/0
Vol. 154,1527-1531.Oetober 1995 Prinkd in U S A
JolnwAL OF UHOLoCY
Copyright 0 1995 by
AMER~CANU R O ~ I C A ASS~CIAT~ON, L INC.
EFFECT OF ANOXIA ON THE URETHRAL RESPONSE TO PHENYLEPHRINE HEON-YOUNG KWON, ALAN J. WEIN
AND
ROBERT M. LEVIN*
From the Division of Urology, University of Pennsylvania, School of Medicine and the Veteran's Medical Center. Philadelphia, Pennsylvania and Dong-A University School of Medicine, Pusan, Korea
ABSTRACT
Purpose: Smooth muscle requires oxygen for proper contractile function. In addition, anoxia has been shown to inhibit the contractile response to various forms of stimulation. Continence, a t least in part, depends upon active a-adrenergic tone, which provides intraurethral tension during bladder filling. The current study was designed to determine the effect of anoxia on the ability of the urethra to sustain tension. Materials and Methods: Standard isolated urethral strip preparations were used to determine the effect of anoxia on the contractile response to phenylephrine; isolated whole urethra preparations were used to determine the effect of anoxia on the intraurethral pressureflow characteristics. Results: The peak tension responses of the female and male urethra to phenylephrine were significantly greater than the plateau tension. Anoxia induced an immediate decrease in phenylephrine-stimulated tonic tension to baseline tension. I n the whole urethra preparation, phenylephrine increased opening pressure to a greater extent in the male urethra than in the female urethra. In both sexes, anoxia reduced the opening pressure and completely prevented phenylephrine stimulated increases in opening pressure. Conclusion: The tonic response to phenylephrine is lost virtually immediately upon the initiation of hypoxia. This suggests that hypoxia decreases the ability of the urethra to maintain a tonic response to a-adrenergic stimulation during bladder filling and may be a factor in incontinence, especially in females. KEYWORDS: urethra, anoxia, phenylephrine The proper functioning of all smooth muscle structures depends on the ability of normal blood to deliver oxygen and nutrients. Any pathological mechanisms that interfere with the normal blood supply would have detrimental effects on smooth muscle function.1.2 Clinically, blood flow to the urethra can be compromised by atherosclerosis,thrombophlebitis, infravesical obstruction and pelvic vessel i n j ~ r y .One ~ . ~of the primary consequences of reduced blood flow to the urethra is hypoxia, with a concomitant reduction in oxidative phosphorylation. Incontinencewill occur if urethral tension is reduced below intravesical pressure. Urethral tension depends on a combination of both structural elements within and around the urethra and active a-adrenergic tone. The urethra in the male is significantly longer than in the female, and the external sphincter and prostate contribute to urethral tension in the male. Thus, a-adrenergic tone would play a more important role in continence in the female than in the male. our previous studies6.6 showed that, as a result of anoxia, the ability of the detrusor smooth muscle to sustain tension in the presence of muscarinic Stimulation (bethanechol)decreased rapidly, while the phasic response decreased gradua y over a 60-minute period. The intracellular A W concentration decreased concomitantly with phasic tension. Upon reoxygenation (after 60 minutes of anoxia),the detrusor rapidly recovered its tonic tension (in the continual presence of bethanechol) whereas intracellular A W concentration returned gradually over 60 minutes. The conclusions from twepted for publication March 8, 1996.
these studies were that the tonic response to muwarinic stimulation depended upon the level of oxidative phosphorylation (use of oxidative energy) whereas the phasic response used ATP as its energy source. The present study was designed to compare the effect of anoxia on the ability of in vitm urethral strips (obtained &om both male and female rabbits) to generate and sustain tension in response to phenylephrine administrationand to correlate these studies with the functional ability of the whole urethra to sustain tension. METHODS
Isolated strip methodology. Male New Zealand white rabbits weighing 2.2 to 2.8 kg. were used for the experimenta. Each rabbit was anesthetized with ketamindxylazine (25 mg. ketamine, 10mg. xylazinelkg. h t r a m d a r l y ) , and surgical anesthesia was maintained with intravenous pentobarbital. For both male and female rabbits the urinary bladder and urethra were exposed through a lower midline incision and the urethra was removed en bloc after separation of the symphysis pubis. The male urethra was dissected free of connective tissue and separated into 2 segments of equal length (proximal and distal segments); each segment was divided into 2 longitudinal strips. The female urethra (from bladder base to external meatus) was divided into 2 longitudinal strips. Each strip was mounted in an individual bath containing 20 ml. Tyrode's solution at 37C and equilibrated for 30 minutes with a gas mixture of 95% 0,. 5% co,. Preliminary studies demonstrated that maximal response to both field stimulation (FS)and phenylephrine was generated with 1 g. resting tension placed on each strip. "hue for all strips, 1 g. of resting tension was placed on each strip. Anoxia was produced by changing the aeration to 95% N and 5% CO,
Requests for reprints: Division of Urology, 3010 Ravdin courtBuildin , Hospital of the University of Pennsylvania, 3400 g c e St., Pkladelphia, Pennsylvania 19104-4283. ' h i s work was supported in art b research grants from the VA.M.C., N.I.H. grants 1 RO 1 D&3366J,RO-3 46162 and Urological Research Associates, University of Pennsylvania. 1627
1528
ANOXIA AND URETHRAL FUNCTION
for 5 to 60minutes. Under oxygenated conditions, phenylephrine was added to the bath (200 pM.1. At 10 minutes, the gas mixture was changed to nitrogen for 5 to 60 minutes. After a specific time period, the gas mixture was changed back to oxygen. Control experiments monitored the tonic response to phenylephrine for 60 minutes. Isolated whole bladder-urethra methodology.7.8 Individual male and female rabbits were anesthetized as given above. The entire bladder and urethra were surgically removed as a unit. The isolated bladder and urethra unit was cut at the level of the ureters. The base and urethra unit was mounted in a 2,000-ml. organ bath as follows: a catheter was inserted into the bladder base and secured with pursestring suture maintaining the opening above the internal sphincter of the urethra. The distal urethra was mounted onto a tube and secured with pursestring suture. The catheter cannulating the bladder base was connected to a pressure transducer via a 3-way stopcock. A saline bottle was connected to the transducer and urethra at a second port in the stopcock. The height of the saline bottle was smoothly increased until flow through urethra began. The opening pressure was defined as the pressure at which flow begins. Bladder flow at opening pressure was monitored for 15minutes at which time the saline bottle was sequentially raised to 5 cm., 10 em. and 15 cm. above opening pressure. Saline flow through the urethra was averaged over 15-minute periods of time. After these measurements, 200 pM. phenylephrine (final concentration) was added, and the effects on opening pressure and flow rates determined as given above. Anoxia was produced and the bladder-urethra incubated for 30 minutes. At this time opening pressure and flow rates were determined as given above. Oxygen tension in selected experiments was monitored with a Gilson Oxygraph (Middleton, Wisconsin). It took 5 minutes of aeration of the 30 ml. bath with nitrogen to reduce the oxygen tension to minimum levels; it took 12 to 14 minutes to reduce the oxygen tension of the 2 1. bath to minimal value. Each physiological determination was performed on preparations of a minimum of 6 individual animals. High energy phosphate experiments. Isolated strips of male and female urethra (similar to those used for physiological experiments) were incubated in oxygen for 30 minutes. At this time phenylephrine was given and at 10 minutes the aeration changed to nitrogen for 5 to 60 minutes. Individual strips of urethra were rapidly h z e n and stored under liquid nitrogen at the following time periods: after 30 minutes of incubation in oxygen; after 5,15,30 and 60minutes of anoxia (in presence of phenylephrine). Three male and 3 female urethras were used for these experiments. Each frozen strip was ground in a mortar precooled in liquid nitrogen with 2.0 ml. 0.625 N perchloric acid. After thawing and centrifugation, the supernatant was neutralized with one-halfvolume of neutralization buffer [3 parts 0.5 M. triethanolamine (pH 7.0) and 2 parts 1.67 M. K&O,]. The precipitated potassium perchlorate was removed by centrifugation, and ATP and creatine phosphate were measured in aliquot by luciferin-luciferase methodology.10 Statistical analysis of the data was performed by analysis of variance followed by multiple comparisons tests.
EFFECT OF PHENYLEPHRINE ON RESPONSE OF URETHRAL STRIPS
m
PEAK RESPONSE
[XI PLATEAU RESPONSE
t
n .
MALE PROXIMAL URETHRA
MALE DISTAL URETHRA
FEMALE URETHRA
Frc. 1. Effect of phenylephrine on urethral strip tension. Each bar represents mean t standard error of betwefn 6 and 8 individual cantly dlfferent from peak repreparations (animals). x = Si sponse (p c0.05); * = significantserent from male proximal and female urethra (p 10.05). should be stressed that these values are normalized per 100 mg. tissue, and since the mass of the male urethra is significantly greater than the mass of the female, the total tension that can be generated by the male urethra would be considerably greater than that of the female. For all segments, in the absence of anoxia, the tonic response to phenylephrine was constant for 60 minutes of continual stimulation (fig. 2). For all segments, anoxia induced an immediate decrease in phenylephrine stimulated tonic tension to baseline or below (fig. 3). In general, each rabbit provides 2 individual strips, and there was a minimum of 6 animals for each study (a minimum of 12 strips). Approximately half of the strips showed significant spontaneous activity. The level of spontaneous activity between strips is variable and could not be averaged. Anoxia resulted in the complete loss of spontaneous activity in all strips of male urethra and no obvious change in the spontaneous activity of strips of female urethra. For all segments, tonic tension recovered rapidly upon reoxygenation (fig. 3). Up to 30 minutes of continuous anoxia, tonic tension recovered to control levels within 5 minutes. At 60 minutes, recovery at 5 minutes was slightly lower in the male proximal and female urethra. Tonic tension in the male distal urethra returned to control levels after 5 minutes of reoxygenation. In the presence of phenylephrine, anoxia stimulated a more rapid hydrolysis of creatinine phosphate (CP) than A m . At 5 minutes, when tonic tension fell to minimum levels, there was only a minor reduction in the concentration
1
MALE DISTAL U R U H R A
RESULTS
The mean mass of the intact female urethras was 0.65 2 0.12g. whereas the mean mass of the male urethras was 2.25 -C 0.22 g. The thickness of the male urethra was visibly greater than that of the female urethra. Phenylephrine rapidly increased tension to peak values in all strips; this was followed by a reduction to a stable tension (tonic or plateau tension). The peak tension responses for all sections were significantly greater than the plateau tensions (fig. 1). It
t 0
t
PHENYLEPHRINE
10
20
30
40
50
60
TIME (min) FIG.2. Representative example of tonic response of urethral strip to 60 minutes’ stimulation by phenylephrine.
1529
ANOXIA AND URETHRAL FUNCTION Effect of phenylephriw and anoxia on opening pressure
FEMALE U R U H R A
Male Control Phenylephrine Anoxia Anoxia
Female
+ Phenyl. Opening pressures are in em.HaO. Significantly Werent fmm control, p <0.05. t Significantly Werant fmm female, p (0.05. ** Significantlydifferent from phenyiephrine. p cO.06.
5.0 2 10.4 2 2.63 2 2.76 2
0.52 1.29.t 0.39' 0.76'.
-t PHENYL~PHRINE NITROGEN
OXVGEN
MALE PROXIMAL URETHRA
PT z
6.87 z 1.04 17.93 2 1.6. 2.13 -t 0.66. 4.4 ? 1.3"
8.0
4.0 -W S
+
OXYGEN
0
10
20
30
40
50
I
60
TIME (min) FIG.3. &presentative tracin s of res me of urethral strips isolated from female and male rab%itato p!&ylephrine, nitrogen (anoxia) and reoxygenation. of ATP. However, by 30 minutes, both ATP and CP were reduced to minimal values (fig. 4). The tonic response to phenylephrine returned rapidly upon reoxygenation, at a time when ATP levels were at minimal values. The opening pressures in the presence and absence of anoxia and phenylephrine are given in the table. Under oxygenated conditions, the opening pressures of the unstimulated urethras were equal for the male and female. Phenylephrine stimulated a significant increase in opening pressure in both male and female urethras, although the magnitude of the phenylephrine-stimulatedincrease in opening pressure was significantly greater for the male as compared with the female urethra (table). Anoxia reduced the opening pressure in the unstimulated male and female urethra to the same extent, and completely prevented the stimulated increase in opening pressure by phenylephrine (table).
EFFECT OF ANOXIA ON HIGH ENERGY PHOSPHATES
The flow rate at opening pressure was significantly greater for the female than for the male, in the presence or absence of phenylephrine (fig. 5). Increases in the pressure above flow rate resulted in similar increases in flow through both the male and female urethra (fig. 6). In conclusion, the tonic response to phenylephrine was rapidly inhibited by anoxia at a time when ATP levels remained high, and fully recovered rapidly upon reoxygenation at a time when ATP levels were reduced below 20% of control values (30 minutes). DISCUSSION
Smooth muscle function requires adequate perfusion with oxygen and nutrients. Ischemia compromises smooth muscle contraction and results in specitic dysfunction of the smooth muscle organ. The extent of the dysfimction depends on the degree and duration of ischemia. Ischemia of the urethra has been the subject of numerous studies.7.11-15 Clinically, the blood supply to the urethra may be compromised by arterial and venous occlusion. The etiologic factors reported in the literature include toxic response to the implantation of indwelling catheters,7 cardiovascular surgery,ll injury, urethroplasty, pelvic thrombophlebitis, chronic vascular insufficiency and, possibly, interstitial cystitis.16 The results of the present study demonstrate that anoxia induces a rapid loss in the tonic response to a-adrenergic stimulation in the female and male urethras. In isolated strips, anoxia resulted in an immediate loss of the tonic response to phenylephrine stimulation. Creatine kinase conversion of CP + ADP to ATP + creatine provides a rapid cytosolic mechanism by which the cyt~solicconcentration of ATP is maintained eonstant during periods of high ATP use (such as during anoxic periods). Creatine kinase activity is responsible for the observation that the concentration of creatine phosphate is reduced to a significantly greater extent
FLOW RATE AT OPENING PRESSURE
0 0 LL
* 60
0 I-
z
W
$
40
20
W
CONTROL
5 rnin
15 rnin
30 min
60 min
DURATION OF ANOXIA RG. 4. Effect of anoxia on high energy phosphates. Each bar represents mean % standard error of mean of 3 of each h o n of
urethra (female, male distal, male p m d a l ) . There were no aignifh u t differences among Merent d o - of m-). * = .sign& -tlY dirrerent from control (p <0.05); x = mpficantly Merent from the concentration of ATP at each time pend (p <0.05).
CONTROL
PHENYLEPHRINE
FIG. 5. Effect of phenylephrine on urethral flow rate at opening pressure of isolated whole urethra preparations. Each bar is mean 5 standard error of mean for 6 individual preparations (animals). * = significantlyWerent from male (p c0.05).
1530
ANOXIA AND URETHRAL FUNCTION
flow at opening pressure was significantly greater for the female than for the male urethra. This is probably due to the absence of the prostate and external sphincter around the 623 MALE URETHRA female urethra. Phenylephrine stimulated a greater increase in opening pressure for the male than for the female urethra. This is consistent with the observation that the male urethra is longer and significantly thicker than the female urethra. However, flow a t opening pressure was not significantly changed in the presence of phenylephrine. The spontaneous activity of urethral strips isolated from female rabbits was not inhibited by anoxia, whereas the spontaneous activity of urethral strips isolated from male rabbits was completely inhibited by anoxia. This would mean that there are significant differences between male and female urethra regarding the generation of spontaneous activity. These differences may be related to the observation that OPENING 5cm 10cm 15cm spontaneous activity of isolated strips of bladder and urethra PRESSURE may depend upon the generation of prostaglandins18-20 and PRESSURE ABOVE OPENING PRESSURE the relationship between estrogen levels and prostaglandin Pressure = cm H20 in the regulation of smooth muscle contractile responses to FIG. 6. Effect of pressure on urethral flow through isolated whole various agonists (myometriud.21.22Further studies are necurethra. Each bar is mean 2 standard error of mean of 6 individual essary to establish the mechanism for these observed differexperiments. * = significantly different from male urethra (p <0.05). ences in spontaneous activity generation in the urethras from male and female rabbits.
EFFECT OF PRESSURE ON URETHRAL FLOW
than that of ATP during the early response to anoxia. The tonic response to phenylephrine was lost in the presence of REFERENCES anoxia at a time when ATP levels were reduced by less than 1. Detar, R.: Mechanism of physiological hypoxia induced depres20%. The tonic response to phenylephrine was restored to sion of vascular smooth muscle contraction. Am. J. Physiol., control levels immediately after reoxygenation, at a time 238: H761, 1980. when high energy phosphate concentrations were at minimal 2. Bozler, E.: Energetics of smooth muscle contraction. In: The values (30minutes). "his indicates that the tonic response to Biochemistry of Smooth Muscle. Edited by N. L. Stephens. a-adrenergic stimulation is metabolically supported by oxiBaltimore: University Park Press, vol. 3,pp. 3-103, 1977. dative-linked energy sources rather than to cytosolic ATP. 3. Dunn, M.: A study of the bladder flow during distension in rabbits. Br. J. Urol., 41: 67, 1975. This is similar to the tonic response of the bladder body to 4. Lapides, J.: Mechanisms of urinary tract infection. Urology, 14: muscarinic stimulation observed in both isolated strips and 217,1971. whole bladder preparations.16.l7 Bladder emptying is a func5. Levin, R. M., High, J. and Wein, A. J.: Metabolic and contractile tion of the ability of the bladder to sustain a contractile effects of anoxia on the rabbit urinary bladder. J. Urol., 128: response to muscarinic stimulation, and anoxia results in an 194,1982. immediate loss in the ability of the in vitro bladder to emp6. Levin, R. M., Brendler, K, Van Arsdalen, K. N. and Wein, A. J.: ty,*6 just as anoxia results in an immediate loss in the ability Functional response of the rabbit urinary bladder to anoxia of the urethra to maintain an increased opening pressure. and ischemia. Neurourol. Urodyn., 2: 233, 1983. The implication of these studies is that continence is re7. Talja, M.,Virtanen, J. and Anderson, L. C.: Toxic catheters and lated to the ability of the urethra to sustain tension, which diminished urethral blood circulation in the induction of urethral strictures. Eur. Urol., 12: 340, 1986. provides the tone required to maintain continence during 8. Chun, A. L.,Gill, H. S., Wein, A. J. and Levin, R. M.: Pharmabladder filling. As expected, in the presence of phenylephcological comparison of the isolated whole urethra model to rine, the opening pressure of the female urethra was signifurethral strip methodology. Pharmacology, 3 9 192, 1989. icantly lower than that of the male urethra and, at opening A. L., Wein, A. J., Gill, H. S. and Levin, R. M.: Response pressure, flow through the female urethra was significantly 9. Chun, of the whole bladder-urethra model (rabbit) to autonomic greater than flow through the male urethra. This observation drugs. Neurourol. Urodyn., 9 165,1990. is consistent with the significantly shorter length and lower 10. Levin, R. M., Ruggieri, M. R., Gill, H. S., Haugaard, N. and Wein, mass of the female urethra as compared with those of the A. J.: Effect of bethanechol on glycolysis and high energy male urethra. phosphate metabolism of the rabbit urinary bladder. J. Urol., 139 646, 1988. The data presented in this report are consistent with the concept that continence depends upon active urethral tone 11. Elhilali, M.M., Hassouna, M., Abdel-Hakim, A. and Teijeira, J.: Urethral stricture following cardiovascular surgery: role of and imply that ischemia can result in an increased incidence urethral ischemia. J. Urol., 135 275, 1986. of incantinence by reducing the ability of the urethra to sustain maximal a-adrenergic tone. The female urethra 12. Mundy, A. R.: Result and complications of urethroplasty and its future. Br. J. Urol., 71: 322, 1993. would be most susceptible to incontinence and to ischemia- 13. Devendra, G., Diokno, A., Davidson, D., Alderson, T., induced decreases in tone, in part because of its significantly Valenzuela, P. and Banerian, K.: Use of artificial sphincter to smaller mass and shorter length, and greater dependence on bowel segment using rectus muscle interposition. Urology, 41: a-adrenergic tone during bladder filling. At this time there is 268, 1993. no clinical support for this theory, perhaps because no one 14. Siroky, M. B., Nephra, A., Vlachiotis, J. and Krane, R. J.: Effect of spinal cord ischemia on vesicourethral function. J. Urol., has studied this possibility. We are currently initiating both 148: 1211, 1992. human and animal studies on this topic. We will use a needle oxygen tension probe to directly correlate the presence of 15. Zhao, Y.,Wein, A. J., Bilgen, A. and Levin, R. M.: The effect of anoxia on in-vitro bladder function. Pharmacology, 4 3 337, incontinence with tissue oxygen tensions. In animals, we are 1992. creating a urethral ischemia to determine whether this re- 16. Irwin, P.and Gallowan, N. T. M.: Impaired bladder perfusion in sult~in incontinence. interstitial cystitis: a study of blood supply using laser Doppler Although the opening pressures (in the absence of pheflowmetry. J. Urol., 149 890. 1993. nylephrine) of the male and female urethra were similar, 17. Bilgen, A..Wein, A. J., Zhao, Y.and Levin, R. M.: The effects of
ANOXIA AND URETHRAL FUNCTION anoxia on the biphasic response of isolated strips of rabbit bladder to field stimulation, bethanechol, methoxamine, and KCI. Pharmacology, 44.283, 1992. 18. Habawam, A.: Roles of prostaglandin on rabbit vesicourethral smooth muscle contraction. Nippon Hinyokika Gakkai Zasshi, 82 1256,1991. 19. Poli, E.,Macaluso, G. and Pozzoli, C.:Actions of two novel prostaglandin analogs, SC-29169and SC-31391,on guinea pig and human isolated urinary bladder. Gen. Pharmacol., 23: 805, 1992. 20. Tamella, T. L. J., Briscoe, J. A. K., Levin, R. M. and Longhurst,
1531
P. A.: Factors underlying the increased sensitivity to field stimulation of urinary bladder strips from streptozotocininduced diabetic rats. Br. J. Urol., 113: 195,1994. 21. Valenzuela, G.J., Germain, A. and Foster, T. C.: Physiology of uterine activity in pregnancy. Cum. Opin. Obetet. Gynecol., 6 640,1993. 22. Schiemann, W.P., Doggwiler, K 0. and Burton, I. L.: Action of adenosine in estrogen-primed nonpregnant guinea pig myometrium: characterization of smooth muscle reeeptor coupling to phosphoinositide metabolism. J. Pharmacol. Exp. "her., W. 429, 1991.
Vol. 154,1527-1531.Oetober 1995 Prinkd in U S A
JolnwAL OF UHOLoCY
Copyright 0 1995 by
AMER~CANU R O ~ I C A ASS~CIAT~ON, L INC.
EFFECT OF ANOXIA ON THE URETHRAL RESPONSE TO PHENYLEPHRINE HEON-YOUNG KWON, ALAN J. WEIN
AND
ROBERT M. LEVIN*
From the Division of Urology, University of Pennsylvania, School of Medicine and the Veteran's Medical Center. Philadelphia, Pennsylvania and Dong-A University School of Medicine, Pusan, Korea
ABSTRACT
Purpose: Smooth muscle requires oxygen for proper contractile function. In addition, anoxia has been shown to inhibit the contractile response to various forms of stimulation. Continence, a t least in part, depends upon active a-adrenergic tone, which provides intraurethral tension during bladder filling. The current study was designed to determine the effect of anoxia on the ability of the urethra to sustain tension. Materials and Methods: Standard isolated urethral strip preparations were used to determine the effect of anoxia on the contractile response to phenylephrine; isolated whole urethra preparations were used to determine the effect of anoxia on the intraurethral pressureflow characteristics. Results: The peak tension responses of the female and male urethra to phenylephrine were significantly greater than the plateau tension. Anoxia induced an immediate decrease in phenylephrine-stimulated tonic tension to baseline tension. I n the whole urethra preparation, phenylephrine increased opening pressure to a greater extent in the male urethra than in the female urethra. In both sexes, anoxia reduced the opening pressure and completely prevented phenylephrine stimulated increases in opening pressure. Conclusion: The tonic response to phenylephrine is lost virtually immediately upon the initiation of hypoxia. This suggests that hypoxia decreases the ability of the urethra to maintain a tonic response to a-adrenergic stimulation during bladder filling and may be a factor in incontinence, especially in females. KEYWORDS: urethra, anoxia, phenylephrine The proper functioning of all smooth muscle structures depends on the ability of normal blood to deliver oxygen and nutrients. Any pathological mechanisms that interfere with the normal blood supply would have detrimental effects on smooth muscle function.1.2 Clinically, blood flow to the urethra can be compromised by atherosclerosis,thrombophlebitis, infravesical obstruction and pelvic vessel i n j ~ r y .One ~ . ~of the primary consequences of reduced blood flow to the urethra is hypoxia, with a concomitant reduction in oxidative phosphorylation. Incontinencewill occur if urethral tension is reduced below intravesical pressure. Urethral tension depends on a combination of both structural elements within and around the urethra and active a-adrenergic tone. The urethra in the male is significantly longer than in the female, and the external sphincter and prostate contribute to urethral tension in the male. Thus, a-adrenergic tone would play a more important role in continence in the female than in the male. our previous studies6.6 showed that, as a result of anoxia, the ability of the detrusor smooth muscle to sustain tension in the presence of muscarinic Stimulation (bethanechol)decreased rapidly, while the phasic response decreased gradua y over a 60-minute period. The intracellular A W concentration decreased concomitantly with phasic tension. Upon reoxygenation (after 60 minutes of anoxia),the detrusor rapidly recovered its tonic tension (in the continual presence of bethanechol) whereas intracellular A W concentration returned gradually over 60 minutes. The conclusions from twepted for publication March 8, 1996.
these studies were that the tonic response to muwarinic stimulation depended upon the level of oxidative phosphorylation (use of oxidative energy) whereas the phasic response used ATP as its energy source. The present study was designed to compare the effect of anoxia on the ability of in vitm urethral strips (obtained &om both male and female rabbits) to generate and sustain tension in response to phenylephrine administrationand to correlate these studies with the functional ability of the whole urethra to sustain tension. METHODS
Isolated strip methodology. Male New Zealand white rabbits weighing 2.2 to 2.8 kg. were used for the experimenta. Each rabbit was anesthetized with ketamindxylazine (25 mg. ketamine, 10mg. xylazinelkg. h t r a m d a r l y ) , and surgical anesthesia was maintained with intravenous pentobarbital. For both male and female rabbits the urinary bladder and urethra were exposed through a lower midline incision and the urethra was removed en bloc after separation of the symphysis pubis. The male urethra was dissected free of connective tissue and separated into 2 segments of equal length (proximal and distal segments); each segment was divided into 2 longitudinal strips. The female urethra (from bladder base to external meatus) was divided into 2 longitudinal strips. Each strip was mounted in an individual bath containing 20 ml. Tyrode's solution at 37C and equilibrated for 30 minutes with a gas mixture of 95% 0,. 5% co,. Preliminary studies demonstrated that maximal response to both field stimulation (FS)and phenylephrine was generated with 1 g. resting tension placed on each strip. "hue for all strips, 1 g. of resting tension was placed on each strip. Anoxia was produced by changing the aeration to 95% N and 5% CO,
Requests for reprints: Division of Urology, 3010 Ravdin courtBuildin , Hospital of the University of Pennsylvania, 3400 g c e St., Pkladelphia, Pennsylvania 19104-4283. ' h i s work was supported in art b research grants from the VA.M.C., N.I.H. grants 1 RO 1 D&3366J,RO-3 46162 and Urological Research Associates, University of Pennsylvania. 1627
1528
ANOXIA AND URETHRAL FUNCTION
for 5 to 60minutes. Under oxygenated conditions, phenylephrine was added to the bath (200 pM.1. At 10 minutes, the gas mixture was changed to nitrogen for 5 to 60 minutes. After a specific time period, the gas mixture was changed back to oxygen. Control experiments monitored the tonic response to phenylephrine for 60 minutes. Isolated whole bladder-urethra methodology.7.8 Individual male and female rabbits were anesthetized as given above. The entire bladder and urethra were surgically removed as a unit. The isolated bladder and urethra unit was cut at the level of the ureters. The base and urethra unit was mounted in a 2,000-ml. organ bath as follows: a catheter was inserted into the bladder base and secured with pursestring suture maintaining the opening above the internal sphincter of the urethra. The distal urethra was mounted onto a tube and secured with pursestring suture. The catheter cannulating the bladder base was connected to a pressure transducer via a 3-way stopcock. A saline bottle was connected to the transducer and urethra at a second port in the stopcock. The height of the saline bottle was smoothly increased until flow through urethra began. The opening pressure was defined as the pressure at which flow begins. Bladder flow at opening pressure was monitored for 15minutes at which time the saline bottle was sequentially raised to 5 cm., 10 em. and 15 cm. above opening pressure. Saline flow through the urethra was averaged over 15-minute periods of time. After these measurements, 200 pM. phenylephrine (final concentration) was added, and the effects on opening pressure and flow rates determined as given above. Anoxia was produced and the bladder-urethra incubated for 30 minutes. At this time opening pressure and flow rates were determined as given above. Oxygen tension in selected experiments was monitored with a Gilson Oxygraph (Middleton, Wisconsin). It took 5 minutes of aeration of the 30 ml. bath with nitrogen to reduce the oxygen tension to minimum levels; it took 12 to 14 minutes to reduce the oxygen tension of the 2 1. bath to minimal value. Each physiological determination was performed on preparations of a minimum of 6 individual animals. High energy phosphate experiments. Isolated strips of male and female urethra (similar to those used for physiological experiments) were incubated in oxygen for 30 minutes. At this time phenylephrine was given and at 10 minutes the aeration changed to nitrogen for 5 to 60 minutes. Individual strips of urethra were rapidly h z e n and stored under liquid nitrogen at the following time periods: after 30 minutes of incubation in oxygen; after 5,15,30 and 60minutes of anoxia (in presence of phenylephrine). Three male and 3 female urethras were used for these experiments. Each frozen strip was ground in a mortar precooled in liquid nitrogen with 2.0 ml. 0.625 N perchloric acid. After thawing and centrifugation, the supernatant was neutralized with one-halfvolume of neutralization buffer [3 parts 0.5 M. triethanolamine (pH 7.0) and 2 parts 1.67 M. K&O,]. The precipitated potassium perchlorate was removed by centrifugation, and ATP and creatine phosphate were measured in aliquot by luciferin-luciferase methodology.10 Statistical analysis of the data was performed by analysis of variance followed by multiple comparisons tests.
EFFECT OF PHENYLEPHRINE ON RESPONSE OF URETHRAL STRIPS
m
PEAK RESPONSE
[XI PLATEAU RESPONSE
t
n .
MALE PROXIMAL URETHRA
MALE DISTAL URETHRA
FEMALE URETHRA
Frc. 1. Effect of phenylephrine on urethral strip tension. Each bar represents mean t standard error of betwefn 6 and 8 individual cantly dlfferent from peak repreparations (animals). x = Si sponse (p c0.05); * = significantserent from male proximal and female urethra (p 10.05). should be stressed that these values are normalized per 100 mg. tissue, and since the mass of the male urethra is significantly greater than the mass of the female, the total tension that can be generated by the male urethra would be considerably greater than that of the female. For all segments, in the absence of anoxia, the tonic response to phenylephrine was constant for 60 minutes of continual stimulation (fig. 2). For all segments, anoxia induced an immediate decrease in phenylephrine stimulated tonic tension to baseline or below (fig. 3). In general, each rabbit provides 2 individual strips, and there was a minimum of 6 animals for each study (a minimum of 12 strips). Approximately half of the strips showed significant spontaneous activity. The level of spontaneous activity between strips is variable and could not be averaged. Anoxia resulted in the complete loss of spontaneous activity in all strips of male urethra and no obvious change in the spontaneous activity of strips of female urethra. For all segments, tonic tension recovered rapidly upon reoxygenation (fig. 3). Up to 30 minutes of continuous anoxia, tonic tension recovered to control levels within 5 minutes. At 60 minutes, recovery at 5 minutes was slightly lower in the male proximal and female urethra. Tonic tension in the male distal urethra returned to control levels after 5 minutes of reoxygenation. In the presence of phenylephrine, anoxia stimulated a more rapid hydrolysis of creatinine phosphate (CP) than A m . At 5 minutes, when tonic tension fell to minimum levels, there was only a minor reduction in the concentration
1
MALE DISTAL U R U H R A
RESULTS
The mean mass of the intact female urethras was 0.65 2 0.12g. whereas the mean mass of the male urethras was 2.25 -C 0.22 g. The thickness of the male urethra was visibly greater than that of the female urethra. Phenylephrine rapidly increased tension to peak values in all strips; this was followed by a reduction to a stable tension (tonic or plateau tension). The peak tension responses for all sections were significantly greater than the plateau tensions (fig. 1). It
t 0
t
PHENYLEPHRINE
10
20
30
40
50
60
TIME (min) FIG.2. Representative example of tonic response of urethral strip to 60 minutes’ stimulation by phenylephrine.
1529
ANOXIA AND URETHRAL FUNCTION Effect of phenylephriw and anoxia on opening pressure
FEMALE U R U H R A
Male Control Phenylephrine Anoxia Anoxia
Female
+ Phenyl. Opening pressures are in em.HaO. Significantly Werent fmm control, p <0.05. t Significantly Werant fmm female, p (0.05. ** Significantlydifferent from phenyiephrine. p cO.06.
5.0 2 10.4 2 2.63 2 2.76 2
0.52 1.29.t 0.39' 0.76'.
-t PHENYL~PHRINE NITROGEN
OXVGEN
MALE PROXIMAL URETHRA
PT z
6.87 z 1.04 17.93 2 1.6. 2.13 -t 0.66. 4.4 ? 1.3"
8.0
4.0 -W S
+
OXYGEN
0
10
20
30
40
50
I
60
TIME (min) FIG.3. &presentative tracin s of res me of urethral strips isolated from female and male rab%itato p!&ylephrine, nitrogen (anoxia) and reoxygenation. of ATP. However, by 30 minutes, both ATP and CP were reduced to minimal values (fig. 4). The tonic response to phenylephrine returned rapidly upon reoxygenation, at a time when ATP levels were at minimal values. The opening pressures in the presence and absence of anoxia and phenylephrine are given in the table. Under oxygenated conditions, the opening pressures of the unstimulated urethras were equal for the male and female. Phenylephrine stimulated a significant increase in opening pressure in both male and female urethras, although the magnitude of the phenylephrine-stimulatedincrease in opening pressure was significantly greater for the male as compared with the female urethra (table). Anoxia reduced the opening pressure in the unstimulated male and female urethra to the same extent, and completely prevented the stimulated increase in opening pressure by phenylephrine (table).
EFFECT OF ANOXIA ON HIGH ENERGY PHOSPHATES
The flow rate at opening pressure was significantly greater for the female than for the male, in the presence or absence of phenylephrine (fig. 5). Increases in the pressure above flow rate resulted in similar increases in flow through both the male and female urethra (fig. 6). In conclusion, the tonic response to phenylephrine was rapidly inhibited by anoxia at a time when ATP levels remained high, and fully recovered rapidly upon reoxygenation at a time when ATP levels were reduced below 20% of control values (30 minutes). DISCUSSION
Smooth muscle function requires adequate perfusion with oxygen and nutrients. Ischemia compromises smooth muscle contraction and results in specitic dysfunction of the smooth muscle organ. The extent of the dysfimction depends on the degree and duration of ischemia. Ischemia of the urethra has been the subject of numerous studies.7.11-15 Clinically, the blood supply to the urethra may be compromised by arterial and venous occlusion. The etiologic factors reported in the literature include toxic response to the implantation of indwelling catheters,7 cardiovascular surgery,ll injury, urethroplasty, pelvic thrombophlebitis, chronic vascular insufficiency and, possibly, interstitial cystitis.16 The results of the present study demonstrate that anoxia induces a rapid loss in the tonic response to a-adrenergic stimulation in the female and male urethras. In isolated strips, anoxia resulted in an immediate loss of the tonic response to phenylephrine stimulation. Creatine kinase conversion of CP + ADP to ATP + creatine provides a rapid cytosolic mechanism by which the cyt~solicconcentration of ATP is maintained eonstant during periods of high ATP use (such as during anoxic periods). Creatine kinase activity is responsible for the observation that the concentration of creatine phosphate is reduced to a significantly greater extent
FLOW RATE AT OPENING PRESSURE
0 0 LL
* 60
0 I-
z
W
$
40
20
W
CONTROL
5 rnin
15 rnin
30 min
60 min
DURATION OF ANOXIA RG. 4. Effect of anoxia on high energy phosphates. Each bar represents mean % standard error of mean of 3 of each h o n of
urethra (female, male distal, male p m d a l ) . There were no aignifh u t differences among Merent d o - of m-). * = .sign& -tlY dirrerent from control (p <0.05); x = mpficantly Merent from the concentration of ATP at each time pend (p <0.05).
CONTROL
PHENYLEPHRINE
FIG. 5. Effect of phenylephrine on urethral flow rate at opening pressure of isolated whole urethra preparations. Each bar is mean 5 standard error of mean for 6 individual preparations (animals). * = significantlyWerent from male (p c0.05).
1530
ANOXIA AND URETHRAL FUNCTION
flow at opening pressure was significantly greater for the female than for the male urethra. This is probably due to the absence of the prostate and external sphincter around the 623 MALE URETHRA female urethra. Phenylephrine stimulated a greater increase in opening pressure for the male than for the female urethra. This is consistent with the observation that the male urethra is longer and significantly thicker than the female urethra. However, flow a t opening pressure was not significantly changed in the presence of phenylephrine. The spontaneous activity of urethral strips isolated from female rabbits was not inhibited by anoxia, whereas the spontaneous activity of urethral strips isolated from male rabbits was completely inhibited by anoxia. This would mean that there are significant differences between male and female urethra regarding the generation of spontaneous activity. These differences may be related to the observation that OPENING 5cm 10cm 15cm spontaneous activity of isolated strips of bladder and urethra PRESSURE may depend upon the generation of prostaglandins18-20 and PRESSURE ABOVE OPENING PRESSURE the relationship between estrogen levels and prostaglandin Pressure = cm H20 in the regulation of smooth muscle contractile responses to FIG. 6. Effect of pressure on urethral flow through isolated whole various agonists (myometriud.21.22Further studies are necurethra. Each bar is mean 2 standard error of mean of 6 individual essary to establish the mechanism for these observed differexperiments. * = significantly different from male urethra (p <0.05). ences in spontaneous activity generation in the urethras from male and female rabbits.
EFFECT OF PRESSURE ON URETHRAL FLOW
than that of ATP during the early response to anoxia. The tonic response to phenylephrine was lost in the presence of REFERENCES anoxia at a time when ATP levels were reduced by less than 1. Detar, R.: Mechanism of physiological hypoxia induced depres20%. The tonic response to phenylephrine was restored to sion of vascular smooth muscle contraction. Am. J. Physiol., control levels immediately after reoxygenation, at a time 238: H761, 1980. when high energy phosphate concentrations were at minimal 2. Bozler, E.: Energetics of smooth muscle contraction. In: The values (30minutes). "his indicates that the tonic response to Biochemistry of Smooth Muscle. Edited by N. L. Stephens. a-adrenergic stimulation is metabolically supported by oxiBaltimore: University Park Press, vol. 3,pp. 3-103, 1977. dative-linked energy sources rather than to cytosolic ATP. 3. Dunn, M.: A study of the bladder flow during distension in rabbits. Br. J. Urol., 41: 67, 1975. This is similar to the tonic response of the bladder body to 4. Lapides, J.: Mechanisms of urinary tract infection. Urology, 14: muscarinic stimulation observed in both isolated strips and 217,1971. whole bladder preparations.16.l7 Bladder emptying is a func5. Levin, R. M., High, J. and Wein, A. J.: Metabolic and contractile tion of the ability of the bladder to sustain a contractile effects of anoxia on the rabbit urinary bladder. J. Urol., 128: response to muscarinic stimulation, and anoxia results in an 194,1982. immediate loss in the ability of the in vitro bladder to emp6. Levin, R. M., Brendler, K, Van Arsdalen, K. N. and Wein, A. J.: ty,*6 just as anoxia results in an immediate loss in the ability Functional response of the rabbit urinary bladder to anoxia of the urethra to maintain an increased opening pressure. and ischemia. Neurourol. Urodyn., 2: 233, 1983. The implication of these studies is that continence is re7. Talja, M.,Virtanen, J. and Anderson, L. C.: Toxic catheters and lated to the ability of the urethra to sustain tension, which diminished urethral blood circulation in the induction of urethral strictures. Eur. Urol., 12: 340, 1986. provides the tone required to maintain continence during 8. Chun, A. L.,Gill, H. S., Wein, A. J. and Levin, R. M.: Pharmabladder filling. As expected, in the presence of phenylephcological comparison of the isolated whole urethra model to rine, the opening pressure of the female urethra was signifurethral strip methodology. Pharmacology, 3 9 192, 1989. icantly lower than that of the male urethra and, at opening A. L., Wein, A. J., Gill, H. S. and Levin, R. M.: Response pressure, flow through the female urethra was significantly 9. Chun, of the whole bladder-urethra model (rabbit) to autonomic greater than flow through the male urethra. This observation drugs. Neurourol. Urodyn., 9 165,1990. is consistent with the significantly shorter length and lower 10. Levin, R. M., Ruggieri, M. R., Gill, H. S., Haugaard, N. and Wein, mass of the female urethra as compared with those of the A. J.: Effect of bethanechol on glycolysis and high energy male urethra. phosphate metabolism of the rabbit urinary bladder. J. Urol., 139 646, 1988. The data presented in this report are consistent with the concept that continence depends upon active urethral tone 11. Elhilali, M.M., Hassouna, M., Abdel-Hakim, A. and Teijeira, J.: Urethral stricture following cardiovascular surgery: role of and imply that ischemia can result in an increased incidence urethral ischemia. J. Urol., 135 275, 1986. of incantinence by reducing the ability of the urethra to sustain maximal a-adrenergic tone. The female urethra 12. Mundy, A. R.: Result and complications of urethroplasty and its future. Br. J. Urol., 71: 322, 1993. would be most susceptible to incontinence and to ischemia- 13. Devendra, G., Diokno, A., Davidson, D., Alderson, T., induced decreases in tone, in part because of its significantly Valenzuela, P. and Banerian, K.: Use of artificial sphincter to smaller mass and shorter length, and greater dependence on bowel segment using rectus muscle interposition. Urology, 41: a-adrenergic tone during bladder filling. At this time there is 268, 1993. no clinical support for this theory, perhaps because no one 14. Siroky, M. B., Nephra, A., Vlachiotis, J. and Krane, R. J.: Effect of spinal cord ischemia on vesicourethral function. J. Urol., has studied this possibility. We are currently initiating both 148: 1211, 1992. human and animal studies on this topic. We will use a needle oxygen tension probe to directly correlate the presence of 15. Zhao, Y.,Wein, A. J., Bilgen, A. and Levin, R. M.: The effect of anoxia on in-vitro bladder function. Pharmacology, 4 3 337, incontinence with tissue oxygen tensions. In animals, we are 1992. creating a urethral ischemia to determine whether this re- 16. Irwin, P.and Gallowan, N. T. M.: Impaired bladder perfusion in sult~in incontinence. interstitial cystitis: a study of blood supply using laser Doppler Although the opening pressures (in the absence of pheflowmetry. J. Urol., 149 890. 1993. nylephrine) of the male and female urethra were similar, 17. Bilgen, A..Wein, A. J., Zhao, Y.and Levin, R. M.: The effects of
ANOXIA AND URETHRAL FUNCTION anoxia on the biphasic response of isolated strips of rabbit bladder to field stimulation, bethanechol, methoxamine, and KCI. Pharmacology, 44.283, 1992. 18. Habawam, A.: Roles of prostaglandin on rabbit vesicourethral smooth muscle contraction. Nippon Hinyokika Gakkai Zasshi, 82 1256,1991. 19. Poli, E.,Macaluso, G. and Pozzoli, C.:Actions of two novel prostaglandin analogs, SC-29169and SC-31391,on guinea pig and human isolated urinary bladder. Gen. Pharmacol., 23: 805, 1992. 20. Tamella, T. L. J., Briscoe, J. A. K., Levin, R. M. and Longhurst,
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P. A.: Factors underlying the increased sensitivity to field stimulation of urinary bladder strips from streptozotocininduced diabetic rats. Br. J. Urol., 113: 195,1994. 21. Valenzuela, G.J., Germain, A. and Foster, T. C.: Physiology of uterine activity in pregnancy. Cum. Opin. Obetet. Gynecol., 6 640,1993. 22. Schiemann, W.P., Doggwiler, K 0. and Burton, I. L.: Action of adenosine in estrogen-primed nonpregnant guinea pig myometrium: characterization of smooth muscle reeeptor coupling to phosphoinositide metabolism. J. Pharmacol. Exp. "her., W. 429, 1991.