- Email: [email protected]
Influence of strip size and location on contractile responses of rat urinary bladder body strips
Gen. Pharmac. Vol. 26, No. 7, pp. 1519-1527, 1995 Copyright © 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved. 0306-3623/95 $9.50 + 0.00
0306-3623(95)00034-8
Pergamon
Influence of Strip Size and Location on Contractile Responses of Rat Urinary Bladder Body Strips PENELOPE
A. LONGHURST,*
R O B E R T E. L E G G E T T
and JANICE
A . K. B R I S C O E
Division of Urology and Department o f Pharmacology, University o f Pennyslvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. [TEL"(215) 662-6660" Fax: (215) 349-5026] (Received 20 December 1994)
Abstract- 1. We investigated the influence of strip length and dorsal or ventral location of rat urinary bladder strips on contractile responsiveness. 2. No differences occurred in the contractile responses of 0.5, 1.0 and 2.0 cm strips to field stimulation, carbachol, ATP, substance P or to KC1 when the data were expressed as either absolute tension or as tension per cross-sectional area. However, correction for strip mass resulted in significant decreases in the contractile responses of the 2.0-cm strips compared with the 0.5-cm strips. 3. No differences occurred in length-tension curves for ventral and dorsal bladder strips, even though the strips from the dorsal surface appeared thinner than those from the ventral surface. 4. Strips from the ventral surface exhibited more variability in response to field stimulation and were less sensitive to atropine pre-treatment than were those from the dorsal surface. They were also less sensitive to the contractile effects of carbachol than dorsal strips. Dorsal and ventral strips were equally responsive to ATP, substance P and KCl. 5. Our data indicate that the contractile responsiveness of rat urinary bladder strips is independent of strip length. Although there are some differences between the cholinergic responsiveness of strips from the ventral and dorsal surfaces of the bladder, the differences are so small that for most studies they will probably have no influence on data interpretation. Key Words: Rat, bladder, contraction, ATP, field stimulation, carbachol, size
INTRODUCTION Strips o f urinary bladder are c o m m o n l y used for experiments determining the neurotransmitters responsible for bladder function, as well as for evaluating the potential efficacy of drugs acting on the lower urinary tract (Burnstock et al., 1972; Downie and Dean, 1977; Bissada and Finkbeiner, 1979; Sj6gren and Andersson, 1979; Maggi et al., 1985; Kachur et al., 1988; Brading and Williams, 1990). The bladder o f the cat or rabbit weighs 2-3 g, and several strips with similar responsiveness can be obtained from closely adjacent areas (Levin et al., 1990). However, the bladder o f the rat is considerably smaller, weighing only 50-150 rag. Therefore, investigators must decide whether to use several different rats to obtain d o s e - or frequency-response curves for comparative purposes in the presence o f different agonists and antagonists or attempt to obtain several strips from the ventral and dorsal regions
*To whom all correspondence should be addressed.
o f the same bladder. A second consideration, therefore, covered by this study was the influence o f strip length on contractile responsiveness, because the shorter the strip, the greater the number o f strips that can be obtained from a single bladder. It is established that the bladder body and base differ physiologically in their innervation and pharmacologically in their responses to neurotransmitters and sensitivity to autonomic antagonists (Evardson and Setekleiv, 1968; Levin and Wein, 1979; Levin et aL, 1980, 1981). However, the possibility that the contractile responsiveness o f strips from dorsal and ventral regions o f the rat bladder also differs has not to our knowledge been investigated nor has the influence o f strip length on contractile responsiveness been reported.
MATERIALS A N D METHODS Animals Male Sprague-Dawley rats (250-300 g) were obtained from Ace Animals Inc. (Boyertown, PA, U.S.A.) 1 wk 1519
1520
Penelope A. Longhurst et al.
before experimentation. They received food and water ad libitum.
Tissue preparation Rats were weighed and anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The urinary bladder was removed at the level of the ureters, blotted and weighed. The bladder was placed in ice-cold Krebs-Henseleit buffer, opened along the lateral edges and strips were cut as described below. The strips were suspended on silk sutures in organ baths containing Kreb's buffer at 37°C, bubbled with 95070 02-507o COs and connected to Grass force displacement transducers (FT.03). Contractile responses were recorded on a Grass Model 7E polygraph. During the first 30 min, strips were washed every 10 min and adjusted to 2 g tension.
Comparison of different-sized strips The bladder was opened along the two lateral aspects to form one large rectangular strip with the bladder base region at the top and bottom of the strip. Two full-length strips were cut to measure approximately 25 × 2 mm. Each strip contained sections of both the dorsal and ventral surfaces of the bladder. Longitudinal strips measuring approximately 20 x 2, 10 x 2 and 5 × 2 mm were then cut randomly from the ventral and dorsal surfaces of each bladder and suspended in organ baths as described above. In Results, the groups are defined according to these original strip lengths: 0.5, 1.0 and 2.0 cm. Strip lengths were re-measured after adjustment to 2 g basal tension, just before starting the first stimulation.
Comparison of strips from dorsal and ventral aspects The bladder was opened along the two lateral aspects to form one large rectangular strip with the bladder base region at the top and bottom of the strip. Single longitudinal strips measuring approximately 10 × 2 mm were cut from the center of the ventral and dorsal surfaces and suspended in organ baths as described above.
Immediately after each switch to calcium-free Krebs, the length of the tissue was increased by 2 mm. Passive (in calcium-free Krebs) and active tension (maximal tension in high-potassium Krebs minus passive tension) was determined at each length.
Dose- and frequency-response studies Strips from separate rats were used for frequencyand dose-response studies at a basal tension of 2 g. Electrical stimulation was effected using a Grass $88 stimulator. Tissues were stimulated through ring platinum electrodes, using 100 V, 0.05 msec duration and 15 sec stimulation. Dose-response curves were constructed non-cumulatively.
Drugs and chemicals The composition of the Krebs-Henseleit buffers was as follows: normal Krebs (in mM) 112.2 NaCI, 4.8 KCI, 2.5 CaC12, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 5.6 dextrose; high-potassium Krebs (in mM) 117.0 KCI, 2.5 CaC12, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 5.6 dextrose. ATP, ¢t,13-methylene ATP (ct[3-mATP), atropine, carbamylcholine (carbachol), substance P and tetrodotoxin (TTX) were obtained from Sigma Chemical Company (St. Louis, MO U.S.A.).
Statistical analysis Data are presented as means +_ SEM. Cross-sectional area was calculated as weight/(length x density). Geometric mean ECso values (antilog of mean dose producing one-half the maximal response) and their 95°7o confidence intervals (CI) were calculated by probit analysis from individual dose-response curves using the method of Fleming et al. (1972). Statistical analysis was done using the Student's t-test for unpaired samples for comparisons of dorsal and ventral strips or the Bonferroni test for comparison of different length strips. P < 0.05 was considered significant. RESULTS
Length-tension experiments
Effects of strip length on bladder strip weight and other characteristics
Length-tension experiments were performed as described by Andersson et al. (1988) and Eika et al. (1992). Briefly, after the 30-min equilibration period, the tissues were stimulated for 3-min periods with high-potassium Krebs followed by 7 min relaxation in calcium-free Krebs. This sequence was repeated twice, allowing a stable response to develop. Then the tissue was shortened to a length giving zero response to stimulation with high-potassium Krebs. Sequences of stimulation and relaxation were then applied to all preparations.
Strips were included in the data calculations if their length after the first 30 min of equilibration fell within the following ranges: 0.5 cm (0.4-0.85 cm), 1.0 cm (0.9-1.5 cm) or 2.0 cm 0.9-2.5 cm). Values for strip weight, length and cross-sectional area are given in Table 1. Incremental increases occurred in strip weight with increasing length (r 2 = 0.6374, n = 47). The length and weight of strips from each group was significantly different from those of the other groups. However, no differences occurred in cross-sectional areas.
1521
Influence of size on bladder strip contractility Table 1. Influence of rat bladder strip length on strip weight, basal tension and cross-sectionalarea Original Strip Lengtha 0.5 cm 1.0 cm 2.0 cm 0.67 _+ 0.03 1.13 + 0.03* 2.13 ± 0.06t (0.40-0.84) (0.99-1.31) (1.90-2.65) t Strip weight (mg) 8.19 ± 0.69 14.50 _+ 1.20" 29.86 ± 2.19 Cross-sectional areac (mm=) 1.23 ± 0.11 1.30 ± 0.11 1.42 __. 0.11 Values are means ± SEM of single strips from 15 or 16 rats with ranges in parentheses, aLength in dish when cutting strip; bLength measured after stretching to 2 g basal tension in calcium-containing Krebs; CCalculatedat actual strip lengthb. *Significantdifference compared with 0.5-cm strips; tSignificant difference compared with 0.5- and 1.0-cm strips at P < 0.05.
Actual strip lengthb (cm)
Effects of strip length on contractile responsiveness The results of length-tension experiments done with strips of differing lengths are shown in Fig. 1. As expected, the shorter strips had steeper passive length-tension relationships than the longer strips, and the active tension relationships followed a similar pattern. Optimal active tension was developed over a fairly broad range and was maximal at approximately the same passive tension, regardless of strip length (0.5 cm: 2.67 _+ 0.82 g; 1.0 cm: 3.45 _+ 1.39 g; 2.0 cm: 3.05 _+ 1.05 g). The absolute contractile responses o f 0.5-, 1.0-, and 2.0-cm bladder strips to field stimulation (Fig. 2A) and carbachol (Fig. 3A) were similar, although the responses o f the 0.5-cm strips were somewhat smaller than those o f the larger strips. Similarly, there were no differences between the contractile responses of strips o f different length after correction for strip cross-sectional area (Figs. 2B and 3B). However, after correction for strip mass, the contractile responses of 2.0-cm strips were
significantly smaller than those o f the shorter strips (Figs. 2C and 3C). Although absolute contractile responses of bladder strips to A T E substance P and KCI appeared to increase with increasing strip length, there were no significant differences between groups (Fig. 4A). Similar to the findings with responses to field stimulation and carbachol, after correction for cross-sectional area, t h e r e were no differences between the contractile responses o f strips of different lengths to 1 m M ATP, 1 ~tM substance P or 120 m M KC1 (Fig. 4B). However, after correction for strip mass, responses of the 2.0-cm strips to ATP, substance P and KCI were significantly smaller than those of shorter strips (Fig. 4C).
Influence of dorsal and ventral strip location on length-tension experiments Despite a'general impression that strips from the dorsal surface were thinner than strips from the ventral
PASSIVE TENSION
ACTIVE TENSION
70 • 60
•
0.5 cm
0
0.5cm
1.0 cm
[3
1.0cm
,~ /
O)
2.0cm
•
50
L~ 2.0 cm
~ 3
40
.i
30
10
0
"
0
-
-
~
T
10
.
.
.
.
~
20
Increase in Length (mm)
.
.
.
o
.
30
.
.
.
.
I
10
.
.
.
.
I
.
.
.
.
20
Increase in Length (mm)
Fig. 1. Length-tension curves for 0.5-, 1.0-, and 2.0-cm length rat bladder body strips. Each point represents the mean + SEM of single strips from six or seven rats.
30
Penelope A. Longhurst et aL
1522 A
7 •
0.5 c m
•
1 .o c m
surface, there were no differences in either the passive or active length-tension relationships for strips from the dorsal or ventral surface of the rat bladder body (Fig. 5).
General characteristics and responses o f ventral and dorsal bladder strips to electrical stimulation and drug application g,
i
I
I
I
i
I
I
I
0.5
1
2
4
8
16
32
64
16
32
64
*
*
Frequency(Hz) •
E E
0.5 c m
•
1.0 cm
•
2.0 c m
3
g I..-
0.5
1
2
4
8
Frequency (Hz)
(:
45 @
0.5 cm
40
•
1.0 cm
35
•
2.0 cm
T
.
~
3O
E oo
25 20
Strips from the ventral surface seemed to respond much faster to drugs and electrical stimulation and were more prone to develop spontaneous activity than those from the dorsal surface. There were no differences in the magnitude of the responses to electrical stimulation (Fig. 6). After incubation with atropine (1 I~M, 15 rain), contractile response decreased (response to 32 Hz: dorsal 58 _ 5o70, ventral 69 _+ 17o70 of no drug maximum). There was considerable variation in the susceptibility of ventral strips to atropine; on one day a strip from one rat was inhibited by 80°70,whereas a strip from a second rat in an adjacent organ bath was stimulated by 22o70. This degree of variability was not seen with dorsal strips. Although there appeared to be a greater suppression of the contractile response of ventral bladder strips by atropine than dorsal strips, the differences were not significant. Subsequent addition of ¢tl3-mATP caused a further reduction in response to 26 _+ 5°7oof the no drug maximum in dorsal strips and 31 _+ 8°7oin ventral strips at 32 Hz. The responses to 32 Hz were reduced to 4 + 2°70 and 11 + 6°70 of no drug maximum, respectively, after incubation with TTX, indicating a neurogenic origin. Carbachol caused dose-dependent increases in tension in both ventral and dorsal strips, with maximal responses occurring at 30 laM (Fig. 7). The responses of dorsal strips to 0.1-3 ~tM carbachol were significantly larger than those of ventral strips. In addition, dorsal strips were significantly more sensitive to carbachol than ventral strips. Carbachol ECso values were 0.55 IxM for dorsal strips (95°7o CI: 0.48-0.62 IxM) and 0.89 ~tM for ventral strips (95070 CI: 0.72-1.12 ~tM). No differences occurred in the magnitude or sensitivity of dorsal and ventral bladder body strips to 1 mM ATP, 1 txM substance P or 120 mM KCI (Fig. 8).
15
DISCUSSION 10 5
0
I 0.5
1----r1
2
-~ ..... ~ 4
8
I
I~
b
16
32
64
Numerous studies identified regional differences in pharmacological and physiological responses of the mammalian urinary bladder, but all compared cranial to caudal, rather than distal to ventral, regions. Edvard-
Frequency (Hz)
Fig. 2. Frequency-response curves for 0.5-, 1.0- and 2.0-cm length rat bladder body strips. Data are expressed as absolute maximal responses (A) or corrected for cross-sectional
area (B) or strip mass (C). Each point represents the mean +_ SEM of single strips from seven or eight rats. *Significant difference compared with 0.5-cm strips at P < 0.05.
A
12
10
A • •
0.5 cm 1.0 cm
•
2.0 cm
T
-
8
T
0.5cm tomb 1.0cm 2.0 cm
AIQ0
I-
i
I
I
7
I
I
6
I
i
5
ATP
4
KCI
Sub P
Carbachol (-log M)
B B
6
10 II
0.5 cm
•
1 cm
•
0.5cm 5
T
2 cm
T
~
1.0cm
mmm 2.0cm
~ 4
E E
E E
6
3
g g
4
2
1
0 AVP 7
6
5
Carbachol (-log M)
C
Sub P
KCI
60
90
70
ATP
4
0.5cm
•
0.Scm
•
2.0 cm
T
T
50
~B
1.0 cm
~ 4O
6O
oE 30
50
.=_o= 40 I,I--
30
20
~
20
10
10 0 0
I
7
I
I
I
6
I
I
Sub P
KCI
5
Catbachol {-logM)
Fig. 3. Dose-response curves to carbachol for 0.5-, 1.0- and 2.0-cm length rat bladder body strips. Data are expressed as absolute responses (A) or corrected for cross-sectional area (B) or strip mass (C). Each point represents the mean + SEM o f single strips from eight rats. *Significant difference compared with 0.5-cm strips; tSignificant difference compared with 0.5- and 1.0-cm strips at P < 0.05. GP 26:7-F
ATP
Fig. 4. Maximal responses of 0.5-, 1.0- and 2.0-cm length rat bladder body strips to ATP, substance P and KC1. Data are expressed as absolute responses (A) or corrected for crosssectional area (B) or strip mass (C). Each bar represents the mean _+ SEM of single strips from eight rats. *Significant difference compared with 0.5-cm strips.
1524
Penelope A. Longhurst et al. 10
8
60 o Dorsal-passive • Dorsal-active E] Ventral-passive • Ventrel-active
D O R S A L RAT BLADDER BODY STRIPS 4
50
• -
.~
.
6
-~
Control
40
•
Atropine
T
•
Atropine+al3-mATP
~
_~
- 30
~.
/I
•
20
l|~q ~I
v
I
T/.L
"1-rx
A
8 2 10
0
0
0
2
4
6
8
10
12
14
16
18
Increase in Length (mm)
Fig. 5. Length-tension curves for dorsal and ventral rat bladder body strips. Each point represents the mean + SEM of
single strips from six rats. sen and Setekleiv (1968) first demonstrated pharmacologically that strips of cat or rabbit detrusor primarily contained ~-adrenergic receptors mediating relaxation, whereas bladder base strips contained a-receptors mediating contraction after stimulation of the hypogastric nerve The regional a- and [3-adrenoceptor distribution in the rabbit bladder was later confirmed using receptor binding methods by Levin and Wein (1979). A more extensive study by Levin et aL (1981) used pharmacological, histochemical and receptor binding techniques to examine autonomic innervation and responsiveness o f the rabbit bladder body during development. They found a gradual increase in adrenergic innervation from birth to 6 wk of age in the rabbit bladder, which correlated with the development of responsiveness to sympathomimetics and increases in a- and ~-adrenoceptor density. The regional density distribution of ~t- and I~-adrenoceptors was confirmed. In contrast, acetylcholinesterase staining did not change with increasing age, and contractile responsiveness to muscarinic agonists and muscarinic receptor density remained the same but somewhat greater in the body than base. Subsequently, Levin et al. (1980) examined contractile responsiveness of strips cut sequentially from the cranial to caudal pole of the rabbit bladder. Maximal contractile responsiveness to the muscarinic agonist, urecholine and to ATP decreased as strips from a more caudal location were used, whereas the contractile responses to the a-adrenergic agonist, methoxamine, increased. Relaxant reponses to the ~-agonist, isoproterenol, were greater in bladder body than in base strips. To our knowledge, no studies have compared the contractile responsiveness of ventral and distal bladder regions. In a histological report using electron microscopy, Gabella and Uvelius (1990) examined female rat blad-
•
T
T
T
•
I
I
I
0.5
I
2
4
8
16
32
64
Frequency (Hz)
VENTRAL RAT BLADDER BODY STRIPS
•
3
Control
•
Atropine
•
Atropine + o.~-mATP
T
T
• TTX
1
0
'~
~-
,
,
,
~
,
0.5
1
2
4
8
16
32
64
Frequency (Hz)
Fig. 6. Frequency-response curves for dorsal (top) and ventral (bottom) rat bladder body strips in the absence and presence of atropine (1 I~M, 15 rain), atropine + a~-mATP (100 I~M, 15 rain) or TTX (1 ~M, 15 min). Each point represents the mean + SEM of single strips from six rats. ders and found that the ventral aspect of the bladder had a large component of longitudinal muscle, whereas the dorsal aspect had a large sheet-like component of circular muscle. Although we used male rats for our study, we have no reason to believe gender differences would change the orientation or general distribution of the muscle fibers. Their findings, therefore, probably explain why the dorsal strips appeared thinner than the ventral strips in our study. Although it might be expected that the longitudinal smooth muscle fibers
Influence of size on bladder strip contractility 12
5
¸
4
¸
1525
*
10
8
g --8
6
I--
4
2
~D~,"
•
Ventral
= r "
7
6
5
Carbachol(-logM)
0
ATP
AVP
SubstanceP
KCI
SEM of single strips from nine rats. *Significant difference compared with ventral strips at P < 0.05.
Fig. 8. Maximal contractile response of dorsal and ventral rat bladder body strips to contractile agents. Each bar represents the mean + SEM of single strips from nine rats. *Significant difference compared with ventral strips at P < 0.05.
of the ventral strips would produce a steeper passive length-tension relationship than the circular smooth muscle fibers of the dorsal strips, this was not the case. Neither did the orientation of the fibers seem to influence the development of active tension. In general, the findings of our study revealed that dorsal and ventral rat bladder strips responded to contractile agents in a similar manner. The only differences we found were an increased responsiveness and sensitivity of dorsal strips to the muscarinic agonist carbachol and inhibition of the neurogenic response by atropine. Whether these changes are related to regional differences in cholinergic innervation or muscarinic receptors is unknown. In addition, the use of dorsal strips seemed to increase sensitivity and reduce variability between preparations. Most studies of bladder innervation concentrated on the identity of the transmitters responsible for functional characteristics, and again, regional localization has been limited to differences in cranial compared with caudal regions of the bladder (Burnstock, 1990). The pioneering studies by Langworthy and co-workers (1940) identified neurons innervating the bladder and other pelvic organs. Nerves enter the bladder in the region of the ureterovesical junction and then follow the blood vessels laterally up into the bladder dome, before curving to the central regions of the bladder. The relative innervation of the ventral or distal surfaces of the bladder is not described. All strips used for our study were taken from the bladder dome, above the ureterovesical junction. However, if nerve bundles enter the bladder at the location of the ureters, there is a possibility that cholinergic innerva-
tion may be denser on the dorsal side of the bladder, and this might explain the more consistent responses of the dorsal strips to field stimulation and susceptibility to atropine. The question remains, does the size of a strip influence the magnitude of the contractile response7 Comparison of the responses of the 0.5- and 1.0-cm strips gave the expected results: doubling the length of the strip resulted in a near doubling of the response to most forms of stimuli, indicating a linear relationship between strip size and length. Normalization of these responses to strip mass confirmed the linear nature of the relationship. However, increasing the length from 1.0 to 2.0 cm resulted in no additional increase in the magnitude of the response, and thus the tension generated per unit mass decreases as the length increases. This may be partially because the 2.0-cm strips were taken from the full length of the bladder and included the dome and both ventral and dorsal portions. Therefore, the likelihood of including muscle fibers running in a non-longitudinalorientation was increased. Conversely, the shorter the strip, and the more circumspect one is about the location from which it is cut, the greater is the likelihood of obtaining strips with consistent fiber orientation and contractile function. These studies, therefore, demonstrate that strip length has to be taken into account and that "normalizing" the response to strip mass does not necessarily make values comparable. In this respect, it is more appropriate to present contractile data in terms of cross-sectional area or as absolute response with strip length and mass reported as well. Invariably, different laboratories select their own parameters for experimentation. However, practically,
Fig. 7. Dose-response curves to carbachol for dorsal and ventral rat bladder body strips. Each point representsthe mean +
1526
Penelope A. Longhurst et aL
strips longer t h a n 1.5-2.0 cm are difficult to work with, because long o r g a n b a t h s are needed. Realistically, the absolute m a g n i t u d e o f the response is u n i m p o r t a n t as long as the o p t i m a l length for the p r e p a r a t i o n is used a n d all c o m p a r i s o n s are m a d e using the same conditions. In this respect, the b l a d d e r is a relatively easy organ to work with, because bladder strips have a rather b r o a d active tension relationship. M a x i m a l active tension can be generated over a greater range o f passive tension t h a n with most other m u s c l e s - a property that is a n absolute r e q u i r e m e n t for efficient b l a d d e r function. This is p r o b a b l y at least partially related to the linear relationship between b l a d d e r distension a n d s m o o t h muscle cell length (Uvelius, 1976; Uvelius a n d Gabella, 1980). It therefore seems t h a t as long as the b l a d d e r strip p r e p a r a t i o n s are prepared consistently within a p r e d e t e r m i n e d length range, contractile responses c a n be expected to b e reproducible a n d comp a r a b l e between experiments. It s h o u l d be n o t e d t h a t these o b s e r v a t i o n s for norm a l b l a d d e r strip p r e p a r a t i o n s c a n n o t necessarily be transposed to preparations where bladder hypertrophy occurs, such as with bladders f r o m diabetic animals (Longhurst a n d Belis, 1986; Longhurst et al., 1990, 1991) or f r o m animals after outlet o b s t r u c t i o n (Levin et al., 1984; G h o n e i m et al., 1986; Kato et al., 1990; M a l m gren et al., 1990). In these conditions, there m a y be differences in the relative concentrations o f connective tissue a n d contractile proteins a n d / o r in the orientat i o n o f the s m o o t h muscle fibres in the bladders, as well as b i o c h e m i c a l changes t h a t m a y have a n i m p a c t o n energy utilization. W i t h o u t a c o m b i n a t i o n o f histological, b i o c h e m i c a l a n d contractile data, it is therefore p r o b a b l y safest to express d a t a in terms o f absolute c o n t r a c t i o n and, in addition, provide some i n f o r m a t i o n o n the cross-sectional area or mass o f the strips. Acknowledgements-This work was supported by United States Public Health Service Grants DK 41610 and 44801.
REFERENCES Andersson P. O., Malmgren A. and Uvelius B. (1988) Cystometrical and in vitro evaluation of urinary bladder function in rats with streptozotocin-induced diabetes. J. Urol. 139, 1359-1362. Bissada N. K. and Finkbeiner A. E. (1979) In vitro action of digitalis on guinea pig detrusor and urethra. Invest. Urol. 17, 1-2. Brading A. E and Williams J. H. (1990) Contractile responses of smooth muscle strips from rat and guinea-pig urinary bladder to transmural stimulation: effects of atropine and or,[i-methylene ATP. Br. J. Pharmac. 99, 493--498. Burnstock G. (1990) Innervation of bladder and bowel. Ciba Foundation Symp. 151, 2-26. Burnstock G., Dumsday B. and Smythe A. (1972) Atropine
resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purine nucleotide. Br. J.. Pharmac. 44, 451-461. Downie J. W. and Dean D. M. (1977) The contribution of cholinergic postganglionic neurotransmission to contractions of rabbit detrusor. J. Pharmac. exp. Ther. 203, 417425. Eika B., Levin R. M. and Longhurst P. A. (1992) Collagen and bladder function in streptozotocin-diabetic rats: effects of insulin and aminoguanidine. J. Urol. 148, 167-172. Evardson P. and Setekleiv J. (1968) Distribution of adrenergic receptors in the urinary bladder of cats, rabbits and guinea-pigs. Acta Pharmac. Toxicol. 26, 437-445. Fleming W. W., Westfall D. P., de la Lande I. S. and Jellett L. B. (1972) Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmac. exp. Ther. 181, 339-345. Gabella G. and Uvelius B. (1990) Urinary bladder of rat: fine structure of normal and hypertrophic musculature. Cell. Tissue Res. 262, 67-79. Ghoneim G. M., Regnier C. H., Biancani P., Johnson L. and Susset J. G. (1986) Effect of vesical outlet obstruction on detrusor contractility and passive properties in rabbits. J. Urol. 135, 1284-1289. Kachur J. E, Peterson J. S., Carter J. P., Rzeszotarski W. J., Hanson R. C. and Noronha-Blob L. (1988) R and S enantiomers of oxybutynin: pharmacological effects in guinea pig bladder and intestine. J. Pharmac. exp. Ther. 247, 867-872. Kato K., Monson E C., Longhurst P. A., Wein A. J., Haugaard N. and Levin R. M. (1990) The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J. Urol. 143, 600-606. Langworthy O. R., Kolb L. C. and Lewis L. G. (1940). The anatomy of the bladder. In: Physiology o f Micturition (Edited by Langworthy O. R., Kolb L. C. and Lewis L. G.), pp. 1-220. Williams & Wilkins Company, Baltimore. Levin R. M. and Wein A. J. (1979) Distribution and function of adrenergic receptors in the urinary bladder of the rabbit. Molec. Pharmacol. 16, 441-448. Levin R. M., Shofer E S. and Wein A. J. (1980) Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmac. exp. Ther. 212, 536-540. Levin R. M., Malkowicz S. B., Jacobowitz D. and Wein A. J. (1981) The ontogeny of the autonomic innervation and contractile response of the rabbit urinary bladder. J. Pharmac. exp. Ther. 219, 250-257. Levin R. M., High J. and Wein A. J. (1984) The effect of shortterm obstruction on urinary bladder function in the rabbit. J. Urol. 132, 789-791. Levin R. M., Longhurst E A., Kato K., McGuire E. J., Elbadawi A. and Wein A. J. (1990) Comparative physiology and pharmacology of the cat and rabbit urinary bladder. J. Urol. 143, 848-852. Longhurst P. A. and Belis J. A. (1986) Abnormalities of rat bladder contractility in streptozotocin-induced diabetes mellitus. J. Pharmac. exp. Ther. 238, 773-777. Longhurst P. A., Kang J., Wein A. J. and Levin R. M. (1990) Length-tension relationship of urinary bladder strips from streptozotocin-diabetic rats. Pharmacology 40, 110-121. Longhurst P. A., Kauer J. and Levin R. M. (1991) The ability of insulin treatment to reverse or prevent the changes in urinary bladder function caused by streptozotocin-induced diabetes mellitus. Gen. Pharmac. 22, 305-311. Maggi C. A., Santicioli P. and Meli A. (1985) Pharmacological evidence for the existence of two compartments in the twitch response to field stimulation of detrusor strips from the rat urinary bladder. J. Auton. Pharmac. 5, 221-230. Malmgren A., Uvelius B., Andersson K.-E. and Andersson P. O. (1990) On the reversibility of functional bladder changes induced by infravesical outflow obstruction in the rat. J. Urol. 143, 1026-1031.
Influence of size on bladder strip contractility Sj6gren C. and Andersson K.-E. (1979) Inhibition of ATPinduced contraction in the guinea-pig urinary bladder in vitro and in vivo. Acta Pharmac. Toxicol. 44, 221-227. Uvelius B. (1976) Isometric and isotonic length-tension relations and variations in cell length in longitudinal smooth
1527
muscle from rabbit urinary bladder. Acta Physiol. Scand. 97, 1-12. Uvelius B. and Gabella G. (1980) Relation between cell length and force production in urinary bladder smooth muscle. Acta Physiol. Scand. 110, 357-365.
0306-3623(95)00034-8
Pergamon
Influence of Strip Size and Location on Contractile Responses of Rat Urinary Bladder Body Strips PENELOPE
A. LONGHURST,*
R O B E R T E. L E G G E T T
and JANICE
A . K. B R I S C O E
Division of Urology and Department o f Pharmacology, University o f Pennyslvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. [TEL"(215) 662-6660" Fax: (215) 349-5026] (Received 20 December 1994)
Abstract- 1. We investigated the influence of strip length and dorsal or ventral location of rat urinary bladder strips on contractile responsiveness. 2. No differences occurred in the contractile responses of 0.5, 1.0 and 2.0 cm strips to field stimulation, carbachol, ATP, substance P or to KC1 when the data were expressed as either absolute tension or as tension per cross-sectional area. However, correction for strip mass resulted in significant decreases in the contractile responses of the 2.0-cm strips compared with the 0.5-cm strips. 3. No differences occurred in length-tension curves for ventral and dorsal bladder strips, even though the strips from the dorsal surface appeared thinner than those from the ventral surface. 4. Strips from the ventral surface exhibited more variability in response to field stimulation and were less sensitive to atropine pre-treatment than were those from the dorsal surface. They were also less sensitive to the contractile effects of carbachol than dorsal strips. Dorsal and ventral strips were equally responsive to ATP, substance P and KCl. 5. Our data indicate that the contractile responsiveness of rat urinary bladder strips is independent of strip length. Although there are some differences between the cholinergic responsiveness of strips from the ventral and dorsal surfaces of the bladder, the differences are so small that for most studies they will probably have no influence on data interpretation. Key Words: Rat, bladder, contraction, ATP, field stimulation, carbachol, size
INTRODUCTION Strips o f urinary bladder are c o m m o n l y used for experiments determining the neurotransmitters responsible for bladder function, as well as for evaluating the potential efficacy of drugs acting on the lower urinary tract (Burnstock et al., 1972; Downie and Dean, 1977; Bissada and Finkbeiner, 1979; Sj6gren and Andersson, 1979; Maggi et al., 1985; Kachur et al., 1988; Brading and Williams, 1990). The bladder o f the cat or rabbit weighs 2-3 g, and several strips with similar responsiveness can be obtained from closely adjacent areas (Levin et al., 1990). However, the bladder o f the rat is considerably smaller, weighing only 50-150 rag. Therefore, investigators must decide whether to use several different rats to obtain d o s e - or frequency-response curves for comparative purposes in the presence o f different agonists and antagonists or attempt to obtain several strips from the ventral and dorsal regions
*To whom all correspondence should be addressed.
o f the same bladder. A second consideration, therefore, covered by this study was the influence o f strip length on contractile responsiveness, because the shorter the strip, the greater the number o f strips that can be obtained from a single bladder. It is established that the bladder body and base differ physiologically in their innervation and pharmacologically in their responses to neurotransmitters and sensitivity to autonomic antagonists (Evardson and Setekleiv, 1968; Levin and Wein, 1979; Levin et aL, 1980, 1981). However, the possibility that the contractile responsiveness o f strips from dorsal and ventral regions o f the rat bladder also differs has not to our knowledge been investigated nor has the influence o f strip length on contractile responsiveness been reported.
MATERIALS A N D METHODS Animals Male Sprague-Dawley rats (250-300 g) were obtained from Ace Animals Inc. (Boyertown, PA, U.S.A.) 1 wk 1519
1520
Penelope A. Longhurst et al.
before experimentation. They received food and water ad libitum.
Tissue preparation Rats were weighed and anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The urinary bladder was removed at the level of the ureters, blotted and weighed. The bladder was placed in ice-cold Krebs-Henseleit buffer, opened along the lateral edges and strips were cut as described below. The strips were suspended on silk sutures in organ baths containing Kreb's buffer at 37°C, bubbled with 95070 02-507o COs and connected to Grass force displacement transducers (FT.03). Contractile responses were recorded on a Grass Model 7E polygraph. During the first 30 min, strips were washed every 10 min and adjusted to 2 g tension.
Comparison of different-sized strips The bladder was opened along the two lateral aspects to form one large rectangular strip with the bladder base region at the top and bottom of the strip. Two full-length strips were cut to measure approximately 25 × 2 mm. Each strip contained sections of both the dorsal and ventral surfaces of the bladder. Longitudinal strips measuring approximately 20 x 2, 10 x 2 and 5 × 2 mm were then cut randomly from the ventral and dorsal surfaces of each bladder and suspended in organ baths as described above. In Results, the groups are defined according to these original strip lengths: 0.5, 1.0 and 2.0 cm. Strip lengths were re-measured after adjustment to 2 g basal tension, just before starting the first stimulation.
Comparison of strips from dorsal and ventral aspects The bladder was opened along the two lateral aspects to form one large rectangular strip with the bladder base region at the top and bottom of the strip. Single longitudinal strips measuring approximately 10 × 2 mm were cut from the center of the ventral and dorsal surfaces and suspended in organ baths as described above.
Immediately after each switch to calcium-free Krebs, the length of the tissue was increased by 2 mm. Passive (in calcium-free Krebs) and active tension (maximal tension in high-potassium Krebs minus passive tension) was determined at each length.
Dose- and frequency-response studies Strips from separate rats were used for frequencyand dose-response studies at a basal tension of 2 g. Electrical stimulation was effected using a Grass $88 stimulator. Tissues were stimulated through ring platinum electrodes, using 100 V, 0.05 msec duration and 15 sec stimulation. Dose-response curves were constructed non-cumulatively.
Drugs and chemicals The composition of the Krebs-Henseleit buffers was as follows: normal Krebs (in mM) 112.2 NaCI, 4.8 KCI, 2.5 CaC12, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 5.6 dextrose; high-potassium Krebs (in mM) 117.0 KCI, 2.5 CaC12, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 5.6 dextrose. ATP, ¢t,13-methylene ATP (ct[3-mATP), atropine, carbamylcholine (carbachol), substance P and tetrodotoxin (TTX) were obtained from Sigma Chemical Company (St. Louis, MO U.S.A.).
Statistical analysis Data are presented as means +_ SEM. Cross-sectional area was calculated as weight/(length x density). Geometric mean ECso values (antilog of mean dose producing one-half the maximal response) and their 95°7o confidence intervals (CI) were calculated by probit analysis from individual dose-response curves using the method of Fleming et al. (1972). Statistical analysis was done using the Student's t-test for unpaired samples for comparisons of dorsal and ventral strips or the Bonferroni test for comparison of different length strips. P < 0.05 was considered significant. RESULTS
Length-tension experiments
Effects of strip length on bladder strip weight and other characteristics
Length-tension experiments were performed as described by Andersson et al. (1988) and Eika et al. (1992). Briefly, after the 30-min equilibration period, the tissues were stimulated for 3-min periods with high-potassium Krebs followed by 7 min relaxation in calcium-free Krebs. This sequence was repeated twice, allowing a stable response to develop. Then the tissue was shortened to a length giving zero response to stimulation with high-potassium Krebs. Sequences of stimulation and relaxation were then applied to all preparations.
Strips were included in the data calculations if their length after the first 30 min of equilibration fell within the following ranges: 0.5 cm (0.4-0.85 cm), 1.0 cm (0.9-1.5 cm) or 2.0 cm 0.9-2.5 cm). Values for strip weight, length and cross-sectional area are given in Table 1. Incremental increases occurred in strip weight with increasing length (r 2 = 0.6374, n = 47). The length and weight of strips from each group was significantly different from those of the other groups. However, no differences occurred in cross-sectional areas.
1521
Influence of size on bladder strip contractility Table 1. Influence of rat bladder strip length on strip weight, basal tension and cross-sectionalarea Original Strip Lengtha 0.5 cm 1.0 cm 2.0 cm 0.67 _+ 0.03 1.13 + 0.03* 2.13 ± 0.06t (0.40-0.84) (0.99-1.31) (1.90-2.65) t Strip weight (mg) 8.19 ± 0.69 14.50 _+ 1.20" 29.86 ± 2.19 Cross-sectional areac (mm=) 1.23 ± 0.11 1.30 ± 0.11 1.42 __. 0.11 Values are means ± SEM of single strips from 15 or 16 rats with ranges in parentheses, aLength in dish when cutting strip; bLength measured after stretching to 2 g basal tension in calcium-containing Krebs; CCalculatedat actual strip lengthb. *Significantdifference compared with 0.5-cm strips; tSignificant difference compared with 0.5- and 1.0-cm strips at P < 0.05.
Actual strip lengthb (cm)
Effects of strip length on contractile responsiveness The results of length-tension experiments done with strips of differing lengths are shown in Fig. 1. As expected, the shorter strips had steeper passive length-tension relationships than the longer strips, and the active tension relationships followed a similar pattern. Optimal active tension was developed over a fairly broad range and was maximal at approximately the same passive tension, regardless of strip length (0.5 cm: 2.67 _+ 0.82 g; 1.0 cm: 3.45 _+ 1.39 g; 2.0 cm: 3.05 _+ 1.05 g). The absolute contractile responses o f 0.5-, 1.0-, and 2.0-cm bladder strips to field stimulation (Fig. 2A) and carbachol (Fig. 3A) were similar, although the responses o f the 0.5-cm strips were somewhat smaller than those o f the larger strips. Similarly, there were no differences between the contractile responses of strips o f different length after correction for strip cross-sectional area (Figs. 2B and 3B). However, after correction for strip mass, the contractile responses of 2.0-cm strips were
significantly smaller than those o f the shorter strips (Figs. 2C and 3C). Although absolute contractile responses of bladder strips to A T E substance P and KCI appeared to increase with increasing strip length, there were no significant differences between groups (Fig. 4A). Similar to the findings with responses to field stimulation and carbachol, after correction for cross-sectional area, t h e r e were no differences between the contractile responses o f strips of different lengths to 1 m M ATP, 1 ~tM substance P or 120 m M KC1 (Fig. 4B). However, after correction for strip mass, responses of the 2.0-cm strips to ATP, substance P and KCI were significantly smaller than those of shorter strips (Fig. 4C).
Influence of dorsal and ventral strip location on length-tension experiments Despite a'general impression that strips from the dorsal surface were thinner than strips from the ventral
PASSIVE TENSION
ACTIVE TENSION
70 • 60
•
0.5 cm
0
0.5cm
1.0 cm
[3
1.0cm
,~ /
O)
2.0cm
•
50
L~ 2.0 cm
~ 3
40
.i
30
10
0
"
0
-
-
~
T
10
.
.
.
.
~
20
Increase in Length (mm)
.
.
.
o
.
30
.
.
.
.
I
10
.
.
.
.
I
.
.
.
.
20
Increase in Length (mm)
Fig. 1. Length-tension curves for 0.5-, 1.0-, and 2.0-cm length rat bladder body strips. Each point represents the mean + SEM of single strips from six or seven rats.
30
Penelope A. Longhurst et aL
1522 A
7 •
0.5 c m
•
1 .o c m
surface, there were no differences in either the passive or active length-tension relationships for strips from the dorsal or ventral surface of the rat bladder body (Fig. 5).
General characteristics and responses o f ventral and dorsal bladder strips to electrical stimulation and drug application g,
i
I
I
I
i
I
I
I
0.5
1
2
4
8
16
32
64
16
32
64
*
*
Frequency(Hz) •
E E
0.5 c m
•
1.0 cm
•
2.0 c m
3
g I..-
0.5
1
2
4
8
Frequency (Hz)
(:
45 @
0.5 cm
40
•
1.0 cm
35
•
2.0 cm
T
.
~
3O
E oo
25 20
Strips from the ventral surface seemed to respond much faster to drugs and electrical stimulation and were more prone to develop spontaneous activity than those from the dorsal surface. There were no differences in the magnitude of the responses to electrical stimulation (Fig. 6). After incubation with atropine (1 I~M, 15 rain), contractile response decreased (response to 32 Hz: dorsal 58 _ 5o70, ventral 69 _+ 17o70 of no drug maximum). There was considerable variation in the susceptibility of ventral strips to atropine; on one day a strip from one rat was inhibited by 80°70,whereas a strip from a second rat in an adjacent organ bath was stimulated by 22o70. This degree of variability was not seen with dorsal strips. Although there appeared to be a greater suppression of the contractile response of ventral bladder strips by atropine than dorsal strips, the differences were not significant. Subsequent addition of ¢tl3-mATP caused a further reduction in response to 26 _+ 5°7oof the no drug maximum in dorsal strips and 31 _+ 8°7oin ventral strips at 32 Hz. The responses to 32 Hz were reduced to 4 + 2°70 and 11 + 6°70 of no drug maximum, respectively, after incubation with TTX, indicating a neurogenic origin. Carbachol caused dose-dependent increases in tension in both ventral and dorsal strips, with maximal responses occurring at 30 laM (Fig. 7). The responses of dorsal strips to 0.1-3 ~tM carbachol were significantly larger than those of ventral strips. In addition, dorsal strips were significantly more sensitive to carbachol than ventral strips. Carbachol ECso values were 0.55 IxM for dorsal strips (95°7o CI: 0.48-0.62 IxM) and 0.89 ~tM for ventral strips (95070 CI: 0.72-1.12 ~tM). No differences occurred in the magnitude or sensitivity of dorsal and ventral bladder body strips to 1 mM ATP, 1 txM substance P or 120 mM KCI (Fig. 8).
15
DISCUSSION 10 5
0
I 0.5
1----r1
2
-~ ..... ~ 4
8
I
I~
b
16
32
64
Numerous studies identified regional differences in pharmacological and physiological responses of the mammalian urinary bladder, but all compared cranial to caudal, rather than distal to ventral, regions. Edvard-
Frequency (Hz)
Fig. 2. Frequency-response curves for 0.5-, 1.0- and 2.0-cm length rat bladder body strips. Data are expressed as absolute maximal responses (A) or corrected for cross-sectional
area (B) or strip mass (C). Each point represents the mean +_ SEM of single strips from seven or eight rats. *Significant difference compared with 0.5-cm strips at P < 0.05.
A
12
10
A • •
0.5 cm 1.0 cm
•
2.0 cm
T
-
8
T
0.5cm tomb 1.0cm 2.0 cm
AIQ0
I-
i
I
I
7
I
I
6
I
i
5
ATP
4
KCI
Sub P
Carbachol (-log M)
B B
6
10 II
0.5 cm
•
1 cm
•
0.5cm 5
T
2 cm
T
~
1.0cm
mmm 2.0cm
~ 4
E E
E E
6
3
g g
4
2
1
0 AVP 7
6
5
Carbachol (-log M)
C
Sub P
KCI
60
90
70
ATP
4
0.5cm
•
0.Scm
•
2.0 cm
T
T
50
~B
1.0 cm
~ 4O
6O
oE 30
50
.=_o= 40 I,I--
30
20
~
20
10
10 0 0
I
7
I
I
I
6
I
I
Sub P
KCI
5
Catbachol {-logM)
Fig. 3. Dose-response curves to carbachol for 0.5-, 1.0- and 2.0-cm length rat bladder body strips. Data are expressed as absolute responses (A) or corrected for cross-sectional area (B) or strip mass (C). Each point represents the mean + SEM o f single strips from eight rats. *Significant difference compared with 0.5-cm strips; tSignificant difference compared with 0.5- and 1.0-cm strips at P < 0.05. GP 26:7-F
ATP
Fig. 4. Maximal responses of 0.5-, 1.0- and 2.0-cm length rat bladder body strips to ATP, substance P and KC1. Data are expressed as absolute responses (A) or corrected for crosssectional area (B) or strip mass (C). Each bar represents the mean _+ SEM of single strips from eight rats. *Significant difference compared with 0.5-cm strips.
1524
Penelope A. Longhurst et al. 10
8
60 o Dorsal-passive • Dorsal-active E] Ventral-passive • Ventrel-active
D O R S A L RAT BLADDER BODY STRIPS 4
50
• -
.~
.
6
-~
Control
40
•
Atropine
T
•
Atropine+al3-mATP
~
_~
- 30
~.
/I
•
20
l|~q ~I
v
I
T/.L
"1-rx
A
8 2 10
0
0
0
2
4
6
8
10
12
14
16
18
Increase in Length (mm)
Fig. 5. Length-tension curves for dorsal and ventral rat bladder body strips. Each point represents the mean + SEM of
single strips from six rats. sen and Setekleiv (1968) first demonstrated pharmacologically that strips of cat or rabbit detrusor primarily contained ~-adrenergic receptors mediating relaxation, whereas bladder base strips contained a-receptors mediating contraction after stimulation of the hypogastric nerve The regional a- and [3-adrenoceptor distribution in the rabbit bladder was later confirmed using receptor binding methods by Levin and Wein (1979). A more extensive study by Levin et aL (1981) used pharmacological, histochemical and receptor binding techniques to examine autonomic innervation and responsiveness o f the rabbit bladder body during development. They found a gradual increase in adrenergic innervation from birth to 6 wk of age in the rabbit bladder, which correlated with the development of responsiveness to sympathomimetics and increases in a- and ~-adrenoceptor density. The regional density distribution of ~t- and I~-adrenoceptors was confirmed. In contrast, acetylcholinesterase staining did not change with increasing age, and contractile responsiveness to muscarinic agonists and muscarinic receptor density remained the same but somewhat greater in the body than base. Subsequently, Levin et al. (1980) examined contractile responsiveness of strips cut sequentially from the cranial to caudal pole of the rabbit bladder. Maximal contractile responsiveness to the muscarinic agonist, urecholine and to ATP decreased as strips from a more caudal location were used, whereas the contractile responses to the a-adrenergic agonist, methoxamine, increased. Relaxant reponses to the ~-agonist, isoproterenol, were greater in bladder body than in base strips. To our knowledge, no studies have compared the contractile responsiveness of ventral and distal bladder regions. In a histological report using electron microscopy, Gabella and Uvelius (1990) examined female rat blad-
•
T
T
T
•
I
I
I
0.5
I
2
4
8
16
32
64
Frequency (Hz)
VENTRAL RAT BLADDER BODY STRIPS
•
3
Control
•
Atropine
•
Atropine + o.~-mATP
T
T
• TTX
1
0
'~
~-
,
,
,
~
,
0.5
1
2
4
8
16
32
64
Frequency (Hz)
Fig. 6. Frequency-response curves for dorsal (top) and ventral (bottom) rat bladder body strips in the absence and presence of atropine (1 I~M, 15 rain), atropine + a~-mATP (100 I~M, 15 rain) or TTX (1 ~M, 15 min). Each point represents the mean + SEM of single strips from six rats. ders and found that the ventral aspect of the bladder had a large component of longitudinal muscle, whereas the dorsal aspect had a large sheet-like component of circular muscle. Although we used male rats for our study, we have no reason to believe gender differences would change the orientation or general distribution of the muscle fibers. Their findings, therefore, probably explain why the dorsal strips appeared thinner than the ventral strips in our study. Although it might be expected that the longitudinal smooth muscle fibers
Influence of size on bladder strip contractility 12
5
¸
4
¸
1525
*
10
8
g --8
6
I--
4
2
~D~,"
•
Ventral
= r "
7
6
5
Carbachol(-logM)
0
ATP
AVP
SubstanceP
KCI
SEM of single strips from nine rats. *Significant difference compared with ventral strips at P < 0.05.
Fig. 8. Maximal contractile response of dorsal and ventral rat bladder body strips to contractile agents. Each bar represents the mean + SEM of single strips from nine rats. *Significant difference compared with ventral strips at P < 0.05.
of the ventral strips would produce a steeper passive length-tension relationship than the circular smooth muscle fibers of the dorsal strips, this was not the case. Neither did the orientation of the fibers seem to influence the development of active tension. In general, the findings of our study revealed that dorsal and ventral rat bladder strips responded to contractile agents in a similar manner. The only differences we found were an increased responsiveness and sensitivity of dorsal strips to the muscarinic agonist carbachol and inhibition of the neurogenic response by atropine. Whether these changes are related to regional differences in cholinergic innervation or muscarinic receptors is unknown. In addition, the use of dorsal strips seemed to increase sensitivity and reduce variability between preparations. Most studies of bladder innervation concentrated on the identity of the transmitters responsible for functional characteristics, and again, regional localization has been limited to differences in cranial compared with caudal regions of the bladder (Burnstock, 1990). The pioneering studies by Langworthy and co-workers (1940) identified neurons innervating the bladder and other pelvic organs. Nerves enter the bladder in the region of the ureterovesical junction and then follow the blood vessels laterally up into the bladder dome, before curving to the central regions of the bladder. The relative innervation of the ventral or distal surfaces of the bladder is not described. All strips used for our study were taken from the bladder dome, above the ureterovesical junction. However, if nerve bundles enter the bladder at the location of the ureters, there is a possibility that cholinergic innerva-
tion may be denser on the dorsal side of the bladder, and this might explain the more consistent responses of the dorsal strips to field stimulation and susceptibility to atropine. The question remains, does the size of a strip influence the magnitude of the contractile response7 Comparison of the responses of the 0.5- and 1.0-cm strips gave the expected results: doubling the length of the strip resulted in a near doubling of the response to most forms of stimuli, indicating a linear relationship between strip size and length. Normalization of these responses to strip mass confirmed the linear nature of the relationship. However, increasing the length from 1.0 to 2.0 cm resulted in no additional increase in the magnitude of the response, and thus the tension generated per unit mass decreases as the length increases. This may be partially because the 2.0-cm strips were taken from the full length of the bladder and included the dome and both ventral and dorsal portions. Therefore, the likelihood of including muscle fibers running in a non-longitudinalorientation was increased. Conversely, the shorter the strip, and the more circumspect one is about the location from which it is cut, the greater is the likelihood of obtaining strips with consistent fiber orientation and contractile function. These studies, therefore, demonstrate that strip length has to be taken into account and that "normalizing" the response to strip mass does not necessarily make values comparable. In this respect, it is more appropriate to present contractile data in terms of cross-sectional area or as absolute response with strip length and mass reported as well. Invariably, different laboratories select their own parameters for experimentation. However, practically,
Fig. 7. Dose-response curves to carbachol for dorsal and ventral rat bladder body strips. Each point representsthe mean +
1526
Penelope A. Longhurst et aL
strips longer t h a n 1.5-2.0 cm are difficult to work with, because long o r g a n b a t h s are needed. Realistically, the absolute m a g n i t u d e o f the response is u n i m p o r t a n t as long as the o p t i m a l length for the p r e p a r a t i o n is used a n d all c o m p a r i s o n s are m a d e using the same conditions. In this respect, the b l a d d e r is a relatively easy organ to work with, because bladder strips have a rather b r o a d active tension relationship. M a x i m a l active tension can be generated over a greater range o f passive tension t h a n with most other m u s c l e s - a property that is a n absolute r e q u i r e m e n t for efficient b l a d d e r function. This is p r o b a b l y at least partially related to the linear relationship between b l a d d e r distension a n d s m o o t h muscle cell length (Uvelius, 1976; Uvelius a n d Gabella, 1980). It therefore seems t h a t as long as the b l a d d e r strip p r e p a r a t i o n s are prepared consistently within a p r e d e t e r m i n e d length range, contractile responses c a n be expected to b e reproducible a n d comp a r a b l e between experiments. It s h o u l d be n o t e d t h a t these o b s e r v a t i o n s for norm a l b l a d d e r strip p r e p a r a t i o n s c a n n o t necessarily be transposed to preparations where bladder hypertrophy occurs, such as with bladders f r o m diabetic animals (Longhurst a n d Belis, 1986; Longhurst et al., 1990, 1991) or f r o m animals after outlet o b s t r u c t i o n (Levin et al., 1984; G h o n e i m et al., 1986; Kato et al., 1990; M a l m gren et al., 1990). In these conditions, there m a y be differences in the relative concentrations o f connective tissue a n d contractile proteins a n d / o r in the orientat i o n o f the s m o o t h muscle fibres in the bladders, as well as b i o c h e m i c a l changes t h a t m a y have a n i m p a c t o n energy utilization. W i t h o u t a c o m b i n a t i o n o f histological, b i o c h e m i c a l a n d contractile data, it is therefore p r o b a b l y safest to express d a t a in terms o f absolute c o n t r a c t i o n and, in addition, provide some i n f o r m a t i o n o n the cross-sectional area or mass o f the strips. Acknowledgements-This work was supported by United States Public Health Service Grants DK 41610 and 44801.
REFERENCES Andersson P. O., Malmgren A. and Uvelius B. (1988) Cystometrical and in vitro evaluation of urinary bladder function in rats with streptozotocin-induced diabetes. J. Urol. 139, 1359-1362. Bissada N. K. and Finkbeiner A. E. (1979) In vitro action of digitalis on guinea pig detrusor and urethra. Invest. Urol. 17, 1-2. Brading A. E and Williams J. H. (1990) Contractile responses of smooth muscle strips from rat and guinea-pig urinary bladder to transmural stimulation: effects of atropine and or,[i-methylene ATP. Br. J. Pharmac. 99, 493--498. Burnstock G. (1990) Innervation of bladder and bowel. Ciba Foundation Symp. 151, 2-26. Burnstock G., Dumsday B. and Smythe A. (1972) Atropine
resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purine nucleotide. Br. J.. Pharmac. 44, 451-461. Downie J. W. and Dean D. M. (1977) The contribution of cholinergic postganglionic neurotransmission to contractions of rabbit detrusor. J. Pharmac. exp. Ther. 203, 417425. Eika B., Levin R. M. and Longhurst P. A. (1992) Collagen and bladder function in streptozotocin-diabetic rats: effects of insulin and aminoguanidine. J. Urol. 148, 167-172. Evardson P. and Setekleiv J. (1968) Distribution of adrenergic receptors in the urinary bladder of cats, rabbits and guinea-pigs. Acta Pharmac. Toxicol. 26, 437-445. Fleming W. W., Westfall D. P., de la Lande I. S. and Jellett L. B. (1972) Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmac. exp. Ther. 181, 339-345. Gabella G. and Uvelius B. (1990) Urinary bladder of rat: fine structure of normal and hypertrophic musculature. Cell. Tissue Res. 262, 67-79. Ghoneim G. M., Regnier C. H., Biancani P., Johnson L. and Susset J. G. (1986) Effect of vesical outlet obstruction on detrusor contractility and passive properties in rabbits. J. Urol. 135, 1284-1289. Kachur J. E, Peterson J. S., Carter J. P., Rzeszotarski W. J., Hanson R. C. and Noronha-Blob L. (1988) R and S enantiomers of oxybutynin: pharmacological effects in guinea pig bladder and intestine. J. Pharmac. exp. Ther. 247, 867-872. Kato K., Monson E C., Longhurst P. A., Wein A. J., Haugaard N. and Levin R. M. (1990) The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J. Urol. 143, 600-606. Langworthy O. R., Kolb L. C. and Lewis L. G. (1940). The anatomy of the bladder. In: Physiology o f Micturition (Edited by Langworthy O. R., Kolb L. C. and Lewis L. G.), pp. 1-220. Williams & Wilkins Company, Baltimore. Levin R. M. and Wein A. J. (1979) Distribution and function of adrenergic receptors in the urinary bladder of the rabbit. Molec. Pharmacol. 16, 441-448. Levin R. M., Shofer E S. and Wein A. J. (1980) Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmac. exp. Ther. 212, 536-540. Levin R. M., Malkowicz S. B., Jacobowitz D. and Wein A. J. (1981) The ontogeny of the autonomic innervation and contractile response of the rabbit urinary bladder. J. Pharmac. exp. Ther. 219, 250-257. Levin R. M., High J. and Wein A. J. (1984) The effect of shortterm obstruction on urinary bladder function in the rabbit. J. Urol. 132, 789-791. Levin R. M., Longhurst E A., Kato K., McGuire E. J., Elbadawi A. and Wein A. J. (1990) Comparative physiology and pharmacology of the cat and rabbit urinary bladder. J. Urol. 143, 848-852. Longhurst P. A. and Belis J. A. (1986) Abnormalities of rat bladder contractility in streptozotocin-induced diabetes mellitus. J. Pharmac. exp. Ther. 238, 773-777. Longhurst P. A., Kang J., Wein A. J. and Levin R. M. (1990) Length-tension relationship of urinary bladder strips from streptozotocin-diabetic rats. Pharmacology 40, 110-121. Longhurst P. A., Kauer J. and Levin R. M. (1991) The ability of insulin treatment to reverse or prevent the changes in urinary bladder function caused by streptozotocin-induced diabetes mellitus. Gen. Pharmac. 22, 305-311. Maggi C. A., Santicioli P. and Meli A. (1985) Pharmacological evidence for the existence of two compartments in the twitch response to field stimulation of detrusor strips from the rat urinary bladder. J. Auton. Pharmac. 5, 221-230. Malmgren A., Uvelius B., Andersson K.-E. and Andersson P. O. (1990) On the reversibility of functional bladder changes induced by infravesical outflow obstruction in the rat. J. Urol. 143, 1026-1031.
Influence of size on bladder strip contractility Sj6gren C. and Andersson K.-E. (1979) Inhibition of ATPinduced contraction in the guinea-pig urinary bladder in vitro and in vivo. Acta Pharmac. Toxicol. 44, 221-227. Uvelius B. (1976) Isometric and isotonic length-tension relations and variations in cell length in longitudinal smooth
1527
muscle from rabbit urinary bladder. Acta Physiol. Scand. 97, 1-12. Uvelius B. and Gabella G. (1980) Relation between cell length and force production in urinary bladder smooth muscle. Acta Physiol. Scand. 110, 357-365.