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The Decompensated Detrusor I: The Effects of Bladder Outlet Obstruction on the Use of Intracellular Calcium Stores
0022-5347/96/1562-0578$03.00/0
Vol. 156,578-581, August 1996 Printed in U.S.A.
”HE JOURNAL OF UROWCY Copyright 0 1996 by AMERICAN UROWICAL ASSOCIATION,INC.
THE DECOMPENSATED DETRUSOR I: THE EFFECTS OF BLADDER OUTLET OBSTRUCTION ON THE USE OF INTRACELLULAR CALCIUM STORES DOROTHEA R O H R M A ” , ROBERT M. LEVIN, JOHN W. DUCKETT AND STEPHEN A. ZDERIC From the Department of Urology, Children’s Hospital of Philadelphia and Division of Urology, University of Pennsylvania, School of Medicine and Veteran’s Medical Center, Philadelphia, Pennsylvania
ABSTRACT
Purpose: As in other smooth muscle groups, extracellular calcium influx as well as the release of calcium from intracellular storage sites or sarcoplasmic reticulum occur in response to receptor stimulation. The relative participation of extracellular influx versus intracellular release has recently been shown to be influenced by developmental stage and obstruction. Partial bladder outlet obstruction results in marked hypertrophy of the bladder and produces alterations in contractile function. To understand better how this contractile dysfunction after outlet obstruction is influenced by intracellular calcium handling we tested the effects of 2 drugs with known effects on the sarcoplasmic reticulum. Materials and Methods: We evaluated ryanodine, which blocks the release of calcium from the sarcoplasmic reticulum, and thapsigargin, which blocks the ability of the sarcoplasmic reticulum to pump cytosolic calcium back into the storage sites. Rabbit bladders were obstructed for Werent periods, after which detrusor muscle strips were harvested and contractile performance was evaluated in the absence and presence of ryanodine and thapsigargin. Results: In the early phases of outlet obstruction the release of intracellular calcium increased significantly. With prolonged obstruction and detrusor decompensation the intracellular storage sites lost the ability to contribute to the generation of contractile force. Conclusions: Alterations in the calcium handling ability of the smooth muscle cell appear to have an important role in the process of decompensation of bladder function in infravesical obstruction. KEY WORDS:bladder, bladder neck obstruction, rabbits, calcium channel blockers
As in other smooth muscle groups, bladder smooth muscle contraction depends on a transient rise in cytosolic free calcium that may be achieved by extracellular influx via gated ion channels in the plasma membrane and release from intracellular storage sites. The existence of receptor operated intracellular calcium stores in the bladder was demonstrated by Mostwin.’ Previously we reported ample evidence to suggest that with normal development there is a progressive increase in the role of intracellular calcium release using physiological, pharmacological and molecular meth0ds.2~ The intracellular calcium storage sites of smooth muscle may be referred to as sarcoplasmic reticulum or calciosomes and they most likely comprise a heterogeneous group of organelles derived from the endoplasmic reticulum. Sarcoplasmic reticulum usually contains a calcium binding protein, an adenosine triphosphate dependent ion pump to reduce cytosolic calcium concentration, an ion channel to allow release and a receptor to allow a signal to trigger the calcium release. This receptor may be for inositol triphosphate. Alternatively increases in cytosolic calcium may trigger the release of calcium from the storage sites, a process referred to as calcium induced calcium release.6.6 In recent years our laboratory efforts have investigated the changing role of the sarcoplasmic reticulum during normal development. We are now interested in the effects of obstruction on the pathways of intracellular calcium release and repeat uptake. In our present study the effects of 2 drugs that affect cytosolic calcium handling were tested. Ryanodine is
known to inhibit the release of calcium stored in the sarcoplasmic reticulum by binding to an ion channel within the wall of the sarcoplasmic reticulum.7 Thapsigargin inhibits calcium repeat uptake by the sarcoplasmic reticulum by inactivating sarcoplasmic endoplasmic reticulum calcium, magnesium-adenosinetriphosphataseon the sarcoplasmic reticulum surface.8 By using these agents alone or in combination we were able to test the relative contribution of the sarcoplasmic reticulum to contractile force generation a t various time points during obstruction. MATERIALS AND METHODS
All studies were done using mature male New Zealand White rabbits and they were approved by the University of Pennsylvania animal use committee. Animals were divided into 5 groups of 4 animals: control, and 1, 7, 14 and 28-day obstruction groups. Sedation with ketamine and xylazine was followed by the administration of sodium pentobarbital to induce deep anesthesia. After shaving and povidone-iodine preparation of the lower abdomen an 8F plastic catheter was inserted into the bladder. A vertical midline incision was made and the urethra was exposed. Care was taken to identify the ureters and the bladder base, and then a 3-zero silk suture was passed around the proximal urethra and tied over the catheter. To maximize uniformity all obstruction procedures were performed by 1investigator (D. R.). All animals received nalbuphine hydrochloride and gentamicin for 48 hours postoperatively. Surgical survival rates were 95%,conSupported in part by grants from the Veterans Administrationand sistent with previous experience. At the predetermined experimental time points the rabbits Grants DK-RO-1-26508, DK-RO-1-33559 and DK-R-2944144 from the National Institutes of Health. were sedated with ketamine and xylazine, followed by the 578
579
INFRAVESICAL OBSTRUCTION AND CALCIUM METABOLISM Bladder weight and maximal response to 150 pmol. bethanechol Bladder Wt. (p.)
Normal bladder Obstruction groups (days):* 1
I 14 28
Max. Response to 150 pM.Bethanechol (m./100 mg. tissue)
1.80 2 0.12
19.35 i 1.97
4.88 6.95 6.09 26.5
13.95 i 1.83 9.02 z 0.98 12.04 i 1.14 2.95 t 0.40
2 1.13
2 0.05 2 0.45 2 5.50
RESULTS
Values represent averages plus or minus standard error of mean of 4 bladders per group. * Statistically significant versus normal bladders (p <0.05 Newman Keuls test).
administration of sodium pentobarbital to induce deep anesthesia. Through the same incision bladders were excised, blotted with dry paper towels and weighed. From each posterior detrusor 4 longitudinal 1 X 1 x 12 mm. muscle strips were prepared. The strips were attached by 3-zero silk sutures to a n organ bath at 1 end and a Grass isometric force transducer at the other end. Transducer output was directed to a polygraph electronically calibrated with known weights. The water jacketed organ baths were maintained at 37C and contained Tyrode’s solution (125 mM. sodium chloride, 2.7 mM. potassium chloride, 1.8 mM. calcium chloride, 0.4 mM. sodium phosphate, 0.5 mM. magnesium chloride X 7 water, 24 mM. sodium hydrogen carbonate and 5.6 mM. dextrose with 95% oxygen/5% carbon dioxide supplied by a bubbling chamber). After equilibration for 1hour a t slack length strips were stretched at increments of 2.5 mm. After waiting for 10 minutes field stimulation was applied (32 Hz., 70 volts) and active tension was measured. Lo was defined as the length at which maximal active tension was measured and all subsequent work was done at Lo. Peak tension was measured in response to field stimulation and 150 pM. bethanechol. Subsequently the buffer was changed to calcium-free Tyrode’s solution containing 1 mM. egtazic acid and the strips were contracted with field stimulation until no further response was noted. They were then challenged with bethanechol until no further increase in tension was noted. At that point calcium was added to the baths in stepwise increments of 1.5
I
mM. in the presence or absence of 40 pM. thapsigargin and/or 80 pM. ryanodine. Previous experiments showed that these doses produced the optimal responses within 20 minutes of incubation. Each experiment was terminated when the addition of further calcium failed to increase tension and this value was judged to be the maximal response.
As in other experimental studies, outlet obstruction induced a n increase in bladder mass at all time points (see table). It was also noted that as bladder mass increased, there was a corresponding decrease in maximal cholinergic response (see table). Bladder weights and contractile performance of obstruction groups were different from control values (p C0.05). While bladder weight and contractile performance at 1, 7 and 14 days did not differ significantly, they were markedly different from these parameters a t 28 days (p <0.05). It is especially important to note that a t 28 days of outlet obstruction severe contractile dysfunction was induced, which allowed this group of 4 bladders t o be characterized as decompensated (less than 50% of baseline contractile force). In contrast, a t 1, 7 and 14 days after outlet obstruction there remained ample but diminished contractile function, implying that there had been at least partial compensation. The relationship between extracellular calcium repletion and force generated was noted for all time points of obstruction. Figure 1,A shows the data normalized for each strip as percent of maximal force generated by that strip. These data show that after obstruction the calcium dose response curves shifted to the left and that with further decompensation there was a turn toward control values. Figure 1, B shows the effects of ryanodine and thapsigargin alone and in combination. When used individually, the agents shifted the dose response curves to the right but not to a statistically significant degree. However, when used in combination, the dose response curve differed substantially from that of the control in shifting markedly to the right. As shown in figure 2, A this effect was markedly enhanced in bladder smooth muscle a t day 1following obstruction. By days 7 and 14 aRer obstruction there remained a significant effect of the ryano-
I
100 -
100 -
80
a t s
80
-
-
c
60-
HP
40-
s
60-
1 4 0 -
2020 -
0 L
o
2
4
calcium [mMl
I
I
I
6
8
10
0
2
4
caldum
6
8
10
[m t +mapsigargin
+ +ryanodine
+thapsigargin+fyanodine -tcontrol
-A-
4 FIG. 1. A, calcium response curve in absence of calcium storage blockers in 4 bladders per group shows influence of different periods of Partial bladder outlet obstruction, B , influence of 40 pM. thapsigargin and 80 pM. ryanodine alone or in combination on calcium response of 4 unobstructed rabbit bladders per group. *, statistically significant versus normal bladder (p <0.05 Newman Keuls test).
INFRAVESICAL OBSTRUCTION
580
l(K1 lZO
J
AND CALCIUM METABOLISM
I 120 , 1
I
120
t---.--t-. j
20
,
----
--4
A
FIG.2. A , influence of 40 pmol. thapsigargin and 80 pmol. ryanodine in combination on calcium responsecunre of 4 rabbit bladders per group obstructed for 1day. B,influence of 40 pM. tha sigargin and 80 pM. ryanodine in combination on calcium response cu,nre of 4 rabbit bladders per group obstructed for 14 days. *, statistic3ly significant versus normal bladder (p c0.05 Newman Keuls test). c,Influence of 40 pM. thapsigargin and 80 pM.ryanodine in combination on calcium response curve of 4 rabbit bladders obstructed for 28 days.
dine and thapsigargin combination on the calcium dose response curves (fig. 2, B ) , although not as pronounced as in figure 2, A. However, by day 28 all 4 bladders were in a decompensated state, and the addition of ryanodine and thapsigargin had no effect on the calcium dose response curve (fig. 2, C). DISCUSSION
The ability of bladder smooth muscle to generate contractile force in response to agonist induced stimulation depends on calcium influx from the extracellular space as well as release from intracellular storage sites, also referred to as the sarcoplasmic reticulum. The use of pharmacological probes to study the relative contributions of these calcium pools to the process of excitation contraction coupling is an important and complementary component of any study examining the mechanisms of bladder compensation and decompensation after outlet obstruction. It is of little use to study bladders harvested at fixed time points after outlet obstruction unless one has also characterized contractile performance. After this has been done one may then search for the molecular markers that correlate best with contractile dysfunction. This study has the benefit of having induced outlet obstruction in a manner that produced a period of initial bladder compensation at days 1,7 and 14. By compensation we refer to the fact that these bladders underwent hypertrophy and a gain in bladder mass but still had preservation of contractile function (greater than 50% of baseline contractile function). In contrast, at 28 days obstruction was severe enough that a state of decompensation developed. It has been our hypothesis that bladder smooth muscle contractile performance after outlet obstruction is highly dependent on a functional sarcoplasmic reticulum. That the sarcoplasmic reticulum is active in bladder smooth muscle has been proved in other studies from our laboratoryz-4 as well as by our current data. The fact that the calcium dose response curve was shifted to the right by the use of thapsigargin and ryanodine is proof that these intracellular storage sites are active. The greatest shift in the calcium dose response curve occurred a t day 1 after obstruction with moderate shifts at days 7 and 14, implying that the sarcoplasmic reticulum was active as these bladders underwent compensatory hypertrophy. In contrast, by day 28 when decompensation had occurred there was little remaining sarcoplasmic reticulum and these agents had a minimal effect if any. We believe that one of several possible molecular explana-
tions for the mechanism of bladder decompensation is failure of the organelle referred to as the sarcoplasmic reticulum. In another study we showed that assays for sarcoplasmic endoplasmic reticulum calcium, magnesium-adenosinetriphosphatase activity using the thapsigargin inhibition technique had a marked loss (80%)of activity in the decompensated versus control group. In the compensated group there was only a 40% decrease in activity. Furthermore, these physiological and biochemical data were corroborated by Western blot analysis.9 These data are similar to the molecular basis for congestive heart failure induced by aortic banding. In animals stratified by physiological analysis there is a solid correlation between loss of sarcoplasmic reticulum activity and ventricular contractile dysfunction.lO-11 We do not expect that there will be 1mechanism to explain the loss of contractile force in bladder decompensation. However, we believe that this study supports our notion that alterations in smooth cell calcium handling ability and loss of the sarcoplasmic reticulum have a n important role in the process of decompensation. This may be partially because in hypertrophy there is an increase in total cell volume, which must in turn be accompanied by a n increase in the sarcoplasmic reticulum. We speculate that as long as these 2 cellular components increase together there will be efficient delivery of calcium t o the filaments with efficient coupling. On the other hand, if cell volume increases dramatically and the sarcoplasmic reticulum fails to keep pace, there will be poor delivery of cytosolic calcium and inefficient coupling. Our current efforts are directed toward understanding this hypothesis at a histological and ultrastructural level. REFERENCES
1. Mostwin, J. L.: Receptor operated intracellular calcium stores in the smooth muscle of the guinea pig bladder. J. Urol., 133:900, 1985. 2. Zderic, S. A., Sillen, U., Liu, G.-H., Snyder, H. McC., 111, Duckett, J. W., Gong, C. and Levin, R. M.: Developmental aspects of excitation contraction coupling of rabbit bladder smooth muscle. J. Urol., part 2, 152: 679, 1994. 3. Zderic, S.A., Gong, C., Hypolite, J. and Levin, R. M.: Developmental aspects of excitation contraction coupling in bladder smooth muscle. In: Muscle, Matrix and Bladder Function. Edited by S.A. Zderic. New York: Plenum Press, pp. 105-116, 1995. 4. Gong, C., Zderic, S. A. and Levin, R. M.: Ontogeny of the ryanodine receptor in rabbit urinary bladder smooth muscle. Molec. Cell. Biochem., 131: 169, 1994.
INFRAVESICAL OBSTRUCTION AND CALCIUM METABOLISM 5. Somlyo, A. P.: Excitation contraction coupling and the ultrastructure of smooth muscle. Circ. Res., 57: 497, 1985. 6. Berridge, M. J., Cheek, T. R., Bennett, D. and Bootman, M. D.: Ryanodine receptors and intracellular signaling. In: Ryanodine Receptors. Edited by V. Sorrentino. CRC Press, in press. 7. Sorrentino, V. and Volpe, P.: Ryanodine receptors: how many, where and why? Trends Pharm. Sci., 1 4 98, 1993. 8. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drebak, B. K., Bjerrum, P. J., Christensen, S. B. and Hanley, M. R.: Thapsigargin, a novel molecular probe for studying intracellular calcium relase and storage. Agents Actions, 27: 17, 1989. 9. Zderic, S. A., Rohrmann, D., Gong, C., Snyder, H. McC., Duckett, J. W., Wein, A. J . and Levin, R. M.: The decompensated de-
581
trusor 11: evidence for loss of sarcoplasmic reticulum function following bladder outlet obstruction in the rabbit. J. Urol., in press. 10. Meuse, A. J., Perralut, C. L. and Mogan, J . P.: Pathophysiology of cardiac hypertrophy and failure of human myocardium: abnormalities of calcium handling. In: Cellular and Molecular Alterations in the Failing Human Heart. Edited by G. Hasenfuss, C. Holubarsch, H. Just and N.R. Alpert. New York: Springer Verlag, 1992. 11. Feldman, A. M., Weinberg, E. O., Ray, P. E. and Lorell, B. H.: Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding. Circ. Res., 73: 184, 1993.
Vol. 156,578-581, August 1996 Printed in U.S.A.
”HE JOURNAL OF UROWCY Copyright 0 1996 by AMERICAN UROWICAL ASSOCIATION,INC.
THE DECOMPENSATED DETRUSOR I: THE EFFECTS OF BLADDER OUTLET OBSTRUCTION ON THE USE OF INTRACELLULAR CALCIUM STORES DOROTHEA R O H R M A ” , ROBERT M. LEVIN, JOHN W. DUCKETT AND STEPHEN A. ZDERIC From the Department of Urology, Children’s Hospital of Philadelphia and Division of Urology, University of Pennsylvania, School of Medicine and Veteran’s Medical Center, Philadelphia, Pennsylvania
ABSTRACT
Purpose: As in other smooth muscle groups, extracellular calcium influx as well as the release of calcium from intracellular storage sites or sarcoplasmic reticulum occur in response to receptor stimulation. The relative participation of extracellular influx versus intracellular release has recently been shown to be influenced by developmental stage and obstruction. Partial bladder outlet obstruction results in marked hypertrophy of the bladder and produces alterations in contractile function. To understand better how this contractile dysfunction after outlet obstruction is influenced by intracellular calcium handling we tested the effects of 2 drugs with known effects on the sarcoplasmic reticulum. Materials and Methods: We evaluated ryanodine, which blocks the release of calcium from the sarcoplasmic reticulum, and thapsigargin, which blocks the ability of the sarcoplasmic reticulum to pump cytosolic calcium back into the storage sites. Rabbit bladders were obstructed for Werent periods, after which detrusor muscle strips were harvested and contractile performance was evaluated in the absence and presence of ryanodine and thapsigargin. Results: In the early phases of outlet obstruction the release of intracellular calcium increased significantly. With prolonged obstruction and detrusor decompensation the intracellular storage sites lost the ability to contribute to the generation of contractile force. Conclusions: Alterations in the calcium handling ability of the smooth muscle cell appear to have an important role in the process of decompensation of bladder function in infravesical obstruction. KEY WORDS:bladder, bladder neck obstruction, rabbits, calcium channel blockers
As in other smooth muscle groups, bladder smooth muscle contraction depends on a transient rise in cytosolic free calcium that may be achieved by extracellular influx via gated ion channels in the plasma membrane and release from intracellular storage sites. The existence of receptor operated intracellular calcium stores in the bladder was demonstrated by Mostwin.’ Previously we reported ample evidence to suggest that with normal development there is a progressive increase in the role of intracellular calcium release using physiological, pharmacological and molecular meth0ds.2~ The intracellular calcium storage sites of smooth muscle may be referred to as sarcoplasmic reticulum or calciosomes and they most likely comprise a heterogeneous group of organelles derived from the endoplasmic reticulum. Sarcoplasmic reticulum usually contains a calcium binding protein, an adenosine triphosphate dependent ion pump to reduce cytosolic calcium concentration, an ion channel to allow release and a receptor to allow a signal to trigger the calcium release. This receptor may be for inositol triphosphate. Alternatively increases in cytosolic calcium may trigger the release of calcium from the storage sites, a process referred to as calcium induced calcium release.6.6 In recent years our laboratory efforts have investigated the changing role of the sarcoplasmic reticulum during normal development. We are now interested in the effects of obstruction on the pathways of intracellular calcium release and repeat uptake. In our present study the effects of 2 drugs that affect cytosolic calcium handling were tested. Ryanodine is
known to inhibit the release of calcium stored in the sarcoplasmic reticulum by binding to an ion channel within the wall of the sarcoplasmic reticulum.7 Thapsigargin inhibits calcium repeat uptake by the sarcoplasmic reticulum by inactivating sarcoplasmic endoplasmic reticulum calcium, magnesium-adenosinetriphosphataseon the sarcoplasmic reticulum surface.8 By using these agents alone or in combination we were able to test the relative contribution of the sarcoplasmic reticulum to contractile force generation a t various time points during obstruction. MATERIALS AND METHODS
All studies were done using mature male New Zealand White rabbits and they were approved by the University of Pennsylvania animal use committee. Animals were divided into 5 groups of 4 animals: control, and 1, 7, 14 and 28-day obstruction groups. Sedation with ketamine and xylazine was followed by the administration of sodium pentobarbital to induce deep anesthesia. After shaving and povidone-iodine preparation of the lower abdomen an 8F plastic catheter was inserted into the bladder. A vertical midline incision was made and the urethra was exposed. Care was taken to identify the ureters and the bladder base, and then a 3-zero silk suture was passed around the proximal urethra and tied over the catheter. To maximize uniformity all obstruction procedures were performed by 1investigator (D. R.). All animals received nalbuphine hydrochloride and gentamicin for 48 hours postoperatively. Surgical survival rates were 95%,conSupported in part by grants from the Veterans Administrationand sistent with previous experience. At the predetermined experimental time points the rabbits Grants DK-RO-1-26508, DK-RO-1-33559 and DK-R-2944144 from the National Institutes of Health. were sedated with ketamine and xylazine, followed by the 578
579
INFRAVESICAL OBSTRUCTION AND CALCIUM METABOLISM Bladder weight and maximal response to 150 pmol. bethanechol Bladder Wt. (p.)
Normal bladder Obstruction groups (days):* 1
I 14 28
Max. Response to 150 pM.Bethanechol (m./100 mg. tissue)
1.80 2 0.12
19.35 i 1.97
4.88 6.95 6.09 26.5
13.95 i 1.83 9.02 z 0.98 12.04 i 1.14 2.95 t 0.40
2 1.13
2 0.05 2 0.45 2 5.50
RESULTS
Values represent averages plus or minus standard error of mean of 4 bladders per group. * Statistically significant versus normal bladders (p <0.05 Newman Keuls test).
administration of sodium pentobarbital to induce deep anesthesia. Through the same incision bladders were excised, blotted with dry paper towels and weighed. From each posterior detrusor 4 longitudinal 1 X 1 x 12 mm. muscle strips were prepared. The strips were attached by 3-zero silk sutures to a n organ bath at 1 end and a Grass isometric force transducer at the other end. Transducer output was directed to a polygraph electronically calibrated with known weights. The water jacketed organ baths were maintained at 37C and contained Tyrode’s solution (125 mM. sodium chloride, 2.7 mM. potassium chloride, 1.8 mM. calcium chloride, 0.4 mM. sodium phosphate, 0.5 mM. magnesium chloride X 7 water, 24 mM. sodium hydrogen carbonate and 5.6 mM. dextrose with 95% oxygen/5% carbon dioxide supplied by a bubbling chamber). After equilibration for 1hour a t slack length strips were stretched at increments of 2.5 mm. After waiting for 10 minutes field stimulation was applied (32 Hz., 70 volts) and active tension was measured. Lo was defined as the length at which maximal active tension was measured and all subsequent work was done at Lo. Peak tension was measured in response to field stimulation and 150 pM. bethanechol. Subsequently the buffer was changed to calcium-free Tyrode’s solution containing 1 mM. egtazic acid and the strips were contracted with field stimulation until no further response was noted. They were then challenged with bethanechol until no further increase in tension was noted. At that point calcium was added to the baths in stepwise increments of 1.5
I
mM. in the presence or absence of 40 pM. thapsigargin and/or 80 pM. ryanodine. Previous experiments showed that these doses produced the optimal responses within 20 minutes of incubation. Each experiment was terminated when the addition of further calcium failed to increase tension and this value was judged to be the maximal response.
As in other experimental studies, outlet obstruction induced a n increase in bladder mass at all time points (see table). It was also noted that as bladder mass increased, there was a corresponding decrease in maximal cholinergic response (see table). Bladder weights and contractile performance of obstruction groups were different from control values (p C0.05). While bladder weight and contractile performance at 1, 7 and 14 days did not differ significantly, they were markedly different from these parameters a t 28 days (p <0.05). It is especially important to note that a t 28 days of outlet obstruction severe contractile dysfunction was induced, which allowed this group of 4 bladders t o be characterized as decompensated (less than 50% of baseline contractile force). In contrast, a t 1, 7 and 14 days after outlet obstruction there remained ample but diminished contractile function, implying that there had been at least partial compensation. The relationship between extracellular calcium repletion and force generated was noted for all time points of obstruction. Figure 1,A shows the data normalized for each strip as percent of maximal force generated by that strip. These data show that after obstruction the calcium dose response curves shifted to the left and that with further decompensation there was a turn toward control values. Figure 1, B shows the effects of ryanodine and thapsigargin alone and in combination. When used individually, the agents shifted the dose response curves to the right but not to a statistically significant degree. However, when used in combination, the dose response curve differed substantially from that of the control in shifting markedly to the right. As shown in figure 2, A this effect was markedly enhanced in bladder smooth muscle a t day 1following obstruction. By days 7 and 14 aRer obstruction there remained a significant effect of the ryano-
I
100 -
100 -
80
a t s
80
-
-
c
60-
HP
40-
s
60-
1 4 0 -
2020 -
0 L
o
2
4
calcium [mMl
I
I
I
6
8
10
0
2
4
caldum
6
8
10
[m t +mapsigargin
+ +ryanodine
+thapsigargin+fyanodine -tcontrol
-A-
4 FIG. 1. A, calcium response curve in absence of calcium storage blockers in 4 bladders per group shows influence of different periods of Partial bladder outlet obstruction, B , influence of 40 pM. thapsigargin and 80 pM. ryanodine alone or in combination on calcium response of 4 unobstructed rabbit bladders per group. *, statistically significant versus normal bladder (p <0.05 Newman Keuls test).
INFRAVESICAL OBSTRUCTION
580
l(K1 lZO
J
AND CALCIUM METABOLISM
I 120 , 1
I
120
t---.--t-. j
20
,
----
--4
A
FIG.2. A , influence of 40 pmol. thapsigargin and 80 pmol. ryanodine in combination on calcium responsecunre of 4 rabbit bladders per group obstructed for 1day. B,influence of 40 pM. tha sigargin and 80 pM. ryanodine in combination on calcium response cu,nre of 4 rabbit bladders per group obstructed for 14 days. *, statistic3ly significant versus normal bladder (p c0.05 Newman Keuls test). c,Influence of 40 pM. thapsigargin and 80 pM.ryanodine in combination on calcium response curve of 4 rabbit bladders obstructed for 28 days.
dine and thapsigargin combination on the calcium dose response curves (fig. 2, B ) , although not as pronounced as in figure 2, A. However, by day 28 all 4 bladders were in a decompensated state, and the addition of ryanodine and thapsigargin had no effect on the calcium dose response curve (fig. 2, C). DISCUSSION
The ability of bladder smooth muscle to generate contractile force in response to agonist induced stimulation depends on calcium influx from the extracellular space as well as release from intracellular storage sites, also referred to as the sarcoplasmic reticulum. The use of pharmacological probes to study the relative contributions of these calcium pools to the process of excitation contraction coupling is an important and complementary component of any study examining the mechanisms of bladder compensation and decompensation after outlet obstruction. It is of little use to study bladders harvested at fixed time points after outlet obstruction unless one has also characterized contractile performance. After this has been done one may then search for the molecular markers that correlate best with contractile dysfunction. This study has the benefit of having induced outlet obstruction in a manner that produced a period of initial bladder compensation at days 1,7 and 14. By compensation we refer to the fact that these bladders underwent hypertrophy and a gain in bladder mass but still had preservation of contractile function (greater than 50% of baseline contractile function). In contrast, at 28 days obstruction was severe enough that a state of decompensation developed. It has been our hypothesis that bladder smooth muscle contractile performance after outlet obstruction is highly dependent on a functional sarcoplasmic reticulum. That the sarcoplasmic reticulum is active in bladder smooth muscle has been proved in other studies from our laboratoryz-4 as well as by our current data. The fact that the calcium dose response curve was shifted to the right by the use of thapsigargin and ryanodine is proof that these intracellular storage sites are active. The greatest shift in the calcium dose response curve occurred a t day 1 after obstruction with moderate shifts at days 7 and 14, implying that the sarcoplasmic reticulum was active as these bladders underwent compensatory hypertrophy. In contrast, by day 28 when decompensation had occurred there was little remaining sarcoplasmic reticulum and these agents had a minimal effect if any. We believe that one of several possible molecular explana-
tions for the mechanism of bladder decompensation is failure of the organelle referred to as the sarcoplasmic reticulum. In another study we showed that assays for sarcoplasmic endoplasmic reticulum calcium, magnesium-adenosinetriphosphatase activity using the thapsigargin inhibition technique had a marked loss (80%)of activity in the decompensated versus control group. In the compensated group there was only a 40% decrease in activity. Furthermore, these physiological and biochemical data were corroborated by Western blot analysis.9 These data are similar to the molecular basis for congestive heart failure induced by aortic banding. In animals stratified by physiological analysis there is a solid correlation between loss of sarcoplasmic reticulum activity and ventricular contractile dysfunction.lO-11 We do not expect that there will be 1mechanism to explain the loss of contractile force in bladder decompensation. However, we believe that this study supports our notion that alterations in smooth cell calcium handling ability and loss of the sarcoplasmic reticulum have a n important role in the process of decompensation. This may be partially because in hypertrophy there is an increase in total cell volume, which must in turn be accompanied by a n increase in the sarcoplasmic reticulum. We speculate that as long as these 2 cellular components increase together there will be efficient delivery of calcium t o the filaments with efficient coupling. On the other hand, if cell volume increases dramatically and the sarcoplasmic reticulum fails to keep pace, there will be poor delivery of cytosolic calcium and inefficient coupling. Our current efforts are directed toward understanding this hypothesis at a histological and ultrastructural level. REFERENCES
1. Mostwin, J. L.: Receptor operated intracellular calcium stores in the smooth muscle of the guinea pig bladder. J. Urol., 133:900, 1985. 2. Zderic, S. A., Sillen, U., Liu, G.-H., Snyder, H. McC., 111, Duckett, J. W., Gong, C. and Levin, R. M.: Developmental aspects of excitation contraction coupling of rabbit bladder smooth muscle. J. Urol., part 2, 152: 679, 1994. 3. Zderic, S.A., Gong, C., Hypolite, J. and Levin, R. M.: Developmental aspects of excitation contraction coupling in bladder smooth muscle. In: Muscle, Matrix and Bladder Function. Edited by S.A. Zderic. New York: Plenum Press, pp. 105-116, 1995. 4. Gong, C., Zderic, S. A. and Levin, R. M.: Ontogeny of the ryanodine receptor in rabbit urinary bladder smooth muscle. Molec. Cell. Biochem., 131: 169, 1994.
INFRAVESICAL OBSTRUCTION AND CALCIUM METABOLISM 5. Somlyo, A. P.: Excitation contraction coupling and the ultrastructure of smooth muscle. Circ. Res., 57: 497, 1985. 6. Berridge, M. J., Cheek, T. R., Bennett, D. and Bootman, M. D.: Ryanodine receptors and intracellular signaling. In: Ryanodine Receptors. Edited by V. Sorrentino. CRC Press, in press. 7. Sorrentino, V. and Volpe, P.: Ryanodine receptors: how many, where and why? Trends Pharm. Sci., 1 4 98, 1993. 8. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drebak, B. K., Bjerrum, P. J., Christensen, S. B. and Hanley, M. R.: Thapsigargin, a novel molecular probe for studying intracellular calcium relase and storage. Agents Actions, 27: 17, 1989. 9. Zderic, S. A., Rohrmann, D., Gong, C., Snyder, H. McC., Duckett, J. W., Wein, A. J . and Levin, R. M.: The decompensated de-
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