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THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS
Temple University School of Medicine, Departments of 1Urology and 2Pharmacology, Philadelphia, Pennsylvania 19140
Submitted 7 January 2003 ; accepted in final form 16 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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denervation; outlet obstruction; urinary diversion
A number of studies have shown that under certain conditions the M2 receptor subtype can contribute to the contractile response. This includes selective alkylation of M3 receptors in an environment of increased intracellular levels of cAMP in the rat urinary bladder (5, 16), guinea pig ileum (10), and trachea (29) or after alkylation without increasing intracellular cAMP levels in other tissues such as the guinea pig gallbladder (2) and colon (22). Other studies of smooth muscle contraction after experimentally induced pathologies, for example in a cat model of experimentally induced esophagitis (25), in the denervated rat bladder (4), and in a model of acute cholecystitis in the guinea pig gallbladder (2), also suggest that the M2 receptor participates in mediation of contraction. In addition, in otherwise normal tissues, the M2 receptor appears to mediate contraction after inhibition of the sarcoplasmic reticulum calcium ATPase, Gq, phosphatidylinositol-specific phospholipase C, phosphatidylcholine-specific phospholipase C, or protein kinase C (PKC; Refs. 2, 25). Additional evidence for an M2 receptor-mediated contractile pathway was demonstrated by the synergistic affects of M2- and M3-selective antagonists for inhibition of bladder contraction in normal bladders treated with thapsigargin and denervated bladders (6).
Our previous studies showed that both bilateral major pelvic ganglion electrocautery (DEN) and spinal cord injury (SCI) in the rat induce bladder hypertrophy and a change in muscarinic receptor subtype mediating bladder contraction from M3 toward M2 (1, 4). To determine whether this change is a result of bladder hypertrophy or denervation, additional experimental pathologies were studied. These include major pelvic ganglion decentralization (MPG-DEC), bladder outlet obstruction (BOO), urinary diversion (DIV), and urinary diversion with denervation (DIV-DEN).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Surgery. Rats (200-250 g female Sprague-Dawley rats from Ace Animals, Boyertown, PA) were anesthetized with 25 µg/kg buprenorphine and 2% isoflurane in oxygen, and a midline incision was made in the lower abdomen. The pelvic plexus was exposed. For bilateral denervation, both the left and right major pelvic ganglion were cauterized with a hand stitching pencil attached to a model SSE 2 solid-state electrosurgery device (Valleylab, Boulder, CO). For sham-operated animals, the plexus was exposed but left intact. For urinary diversions, both ureters were dissected free, cut, and sutured into the colon. For BOO, the urethra was exposed, a 21-gauge syringe needle was placed parallel to the urethra, a 3-0 silk suture was tied around both the needle and urethra, and the needle was then removed. For MPG-DEC, the nerve fibers entering the ganglion from the spinal cord were severed. The subcutaneous tissue, muscle, and skin were sutured. After surgery, urine was expressed with manual pressure on the lower abdomen twice daily for 3 days.
Immediately before bladder harvesting of the MPG-DEC group, they were tested to ensure that spinal stimulation was ineffective in inducing a bladder contraction while MPG stimulation caused an increase in bladder pressure. For this determination, the bladder was catheterized per urethra with PE-50 tubing connected to a pressure transducer. The bladder was emptied and filled manually until an intravesical pressure of 5 cmH2O was induced. A unipolar pith electrode grounded to the abdomen was inserted into the spinal column between the L2 and L3 vertebral bodies and stimulated with a 2- to 5-s train of square wave pulses at 2 V, 30 Hz, 1-ms duration delivered by a Grass S-88 stimulator (Astro-Med, West Warwick, RI). The major pelvic ganglion was stimulated with bipolar electrodes separated by 4 mm with the same stimulation parameters. These stimulations consistently induced a marked bladder contraction in neurally intact animals. Any MPG-DEC animals that showed a bladder contraction to the spinal stimulation or did not show a bladder contraction to the MPG stimulation were not used.
Muscle strips. Urinary bladders were removed from rats euthanized
by decapitation. The urinary bladder body (tissue above the ureteral orifices)
was dissected free of the serosa and surrounding fat. The bladder was divided
in the midsagittal plane, then cut into longitudinal smooth muscle strips
(
4 x 10 mm). The muscle strips were then suspended with 1 g of
tension in tissue baths containing 15 ml of modified Tyrode solution (125 mM
NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.8 mM
CaCl2, 0.5 mM MgCl2, 23.8 mM NaHCO3, and 5.6
mM glucose) and equilibrated with 95 O2-5% CO2 at
37°C.
Carbachol dose response. After equilibration to the bath solution for 30 min, bladder strips were incubated for 30 min in the presence or absence of antagonist. Dose-response curves were derived from the peak tension developed after cumulative addition of carbachol (10 nM to 300 µM final bath concentration). Only one concentration of antagonist was used for each muscle strip (n = 6-8 strips per antagonist concentration). Dose ratios were determined based on the average of the responses of antagonist free strips. An EC50 value was determined for each strip using a sigmoidal curve fit of the data (Origin, Originlab Northampton, MA). The EC50 values determined in the presence of antagonist were used to generate Schild plots to calculate pA2 values for each antagonist. If the slope of the Schild plot was not significantly different from unity (95% confidence interval), the slope of the Schild plot was constrained to unity to calculate the pKb value. To construct the Schild plot for methoctramine and p-F-HHSiD, doses of 0.3 and 3.0 µM were used. The Schild plot for 4-diphenacetoxy-N-methylpiperidine methiodide (4-DAMP) was done using 3.0, 10.0, and 30.0 nM. Because higher doses of darifenacin appeared unsurmountable and lower doses did not produce a significant shift in the concentration-response curve, a single dose of 30 nM darifenacin was used. The estimated pKb for darifenacin was calculated using the formula pKb = -[log(darifenacin concentration) - log(dose ratio - 1)].
Immunoprecipitations. Immunoprecipitations were performed using antibodies as previously described. (4, 30). Protein concentration in the solubilized receptor preparation was determined by a dye binding assay (Bio-Rad). Muscarinic receptor density is reported as mean ± SE femtomoles per milligram protein in this solubilized receptor preparation. Total muscarinic receptor levels were determined by desalting over Sephadex G-50 minicolumns. At least four determinations were performed on two different pools of bladders for all groups.
Statistical and data analysis. For Fig. 1, contractile force is presented as absolute millinewtons of force generated. Tension displayed in Fig. 2, B and C, is normalized to cross-sectional area defined as weight divided by length (which assumes a tissue density of 1). Statistical analysis of multiple group comparison was performed by ANOVA with a post hoc Newman-Keuls test or a Student's t-test where appropriate (GB-STAT, Dynamic Microsystems, Silver Spring, MD). Statistically significant differences in the affinity values and departure from unity in the slopes derived from the Schild plots were determined using the 95% confidence intervals.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Neither DIV nor DIV-DEN induces hypertrophy as evidenced by an increase in bladder weight-to-body weight ratio compared with sham-operated controls; however, DEN, BOO, and MPG-DEC induce significant hypertrophy by 3 days postsurgery. Interestingly, DIV bladders are significantly smaller than sham-operated or DIV-DEN bladders (Fig. 2A).
EFS responses. To determine the effect of short-term hypertrophy on EFS contractile force, the contractile force normalized to cross-sectional area was determined for each group. Figure 2B shows that no differences in the EFS contraction to a submaximal stimulation of 8 V, 30 Hz, 1 ms is seen with DIV, BOO, or MPG-DEC compared with sham-operated control bladders. DIV-DEN and DEN bladders contract less to EFS than sham-operated controls. Unexpectedly, DIV-DEN bladders contract greater than DEN bladders to EFS, suggesting that the nerve terminals in these bladders are not completely degenerated at 3 days (Fig. 2B).
Carbachol responses. The carbachol-induced maximal contraction (Fig. 2C) is not different between any groups except that the BOO group contracts significantly greater than every other group. As the nerves degenerate or are unable to induce a maximal contraction, the ratio of the carbachol maximum contractile response to the EFS contractile response will increase. This can be used as a measure of functional denervation. DEN, DIV-DEN, MPG-DEC, and BOO bladders are functionally denervated compared with sham-operated bladders. DEN bladders are significantly more functionally denervated than the DIV-DEN, BOO, and MPG-DEC bladders. DIV-DEN bladders are functionally denervated similar to BOO, both of which are more functionally denervated than the MPG-DEC bladders. Comparing the carbachol potency between the groups, a lower carbachol EC50 is found in the DEN bladders compared with sham operated (Fig. 3).
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Muscarinic receptor-subtype protein density. Total, M2,
and M3 receptor protein density was determined by subtype-selective
immunoprecipitation (Fig. 4).
Sham-operated bladders have a significantly different total receptor density
(470 fmol/mg solubilized protein) than every group, with an
M2-to-M3 ratio of 6:1. Total muscarinic receptor
density in the DIV and DIV-DEN bladders is significantly less than sham
operated with M2-to-M3 ratios of about 9:1 and 10:1,
respectively. DEN, BOO, and MPG-DEC bladders have greater total receptor
densities than sham-operated with M2-to-M3 ratios of
about 34:1, 24:1, and 11:1, respectively.
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The hypertrophied bladders (DEN, BOO) have an increase in M2 receptors, a decrease in M3 receptors, and an increase in total receptor density, while the hypertrophied MPG-DEC bladders have an increase in M2 and total receptors, with no change in M3 receptors compared with sham operated. The atrophied bladders (DIV) have a decrease in both M2 and M3 receptors, which is reflected in a decrease in total receptor density. The DIV-DEN bladders, which are neither hypertrophied nor atrophied, have a decrease in M2, M3, and total receptor density.
Correlation of muscarinic receptor-subtype density with functional denervation: As can be seen in Fig. 5, the density of the M2 receptor protein correlates (R = 0.77, P = 0.05) with the degree of functional denervation. However, there is no correlation between the density of the M3 subtype and the degree of functional denervation.
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Affinity of antagonists for inhibition of carbachol-induced contractions. Based on the affinity of a series of muscarinic receptor antagonists for inhibition of carbachol-induced contractions, sham-operated bladder contractions are mediated by the M3 receptor subtype (Table 1). This is based on a high affinity for the M3-selective antagonists p-F-HHSiD (7.7 ± 0.2), 4-DAMP (9.1 ± 0.2), and darifenacin (8.5 ± 0.1) and a low affinity for the M2-selective antagonist methoctramine (6.2 ± 0.2). The affinities in DIV and DIV-DEN are also consistent with M3 receptors mediating contraction. The M3-selective antagonists have a lower affinity in the hypertrophied bladders (DEN, MPGDEC, and BOO), which is consistent with participation of the M2 receptor in mediation of contraction. Paradoxically, the M2-selective antagonist methoctramine has a low affinity for inhibition of contraction in all groups, which seems to preclude participation of M2 receptors in contraction. This phenomenon was also seen in our earlier studies (1, 4), which prompted speculation of the existence of two contractile pathways, one mediated by the M3 receptor subtype, the other mediated by the M2 subtype. Further evidence for the existence of two pathways was provided by studies in which antagonists showed superadditive inhibitory effects in blocking bladder contraction (6).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The only group with denervation-induced supersensitivity is the DEN group, not the MPG-DEC or the DIV-DEN groups (Fig. 3). While the DIV-DEN group does not have increased bladder pressure because of the diversion, the MPG-DEC does. However, the MPGDEC group has intact innervation from the major pelvic ganglion to the bladder. This suggests that, for at least the 3-day postoperative time point, both a complete absence of nerve terminals and increased pressure with concomitant hypertrophy are required for development of carbachol supersensitivity. Muscarinic agonist supersensitivity after 1-3 wk of urinary diversion or MPG decentralization has been reported (12), but we found that this does not occur at the 3-day time point. Our results are somewhat different from previously reported effects of bladder hypertrophy for 1 wk where no increase in the methacholine-induced contraction was seen but supersensitivity to methacholine was noted. These differences may be attributed to the duration of hypertrophy or possibly the method of inducing the hypertrophy [urethral ligature used here vs. paraffin injected into the lumen of the bladder used by Ekstrom et al. (13)]. The duration of the hypertrophy is more likely the reason for the differences, because the supersensitivity was transient and not present 4 wk after paraffin injection (13).
Analysis of the density of muscarinic receptors reveals an increase in total receptor density in the hypertrophied bladders regardless of whether they are denervated. All of this increase is accounted for by an increase in the M2 subtype. All experimental groups except MPG-DEC have a significantly lower density of the M3 subtype. The nonhypertrophied groups (DIV and DIV-DEN) have a decrease in total, M2, and M3 receptor density. Therefore, hypertrophy leads to an increase in density of total and M2 receptors while urinary diversion results in a decrease in density of total and M2 receptors regardless of whether the diversion is accompanied by tissue atrophy (DIV group) or not (DIV-DEN group). Our results are consistent with those reported by Nilvebrant et al. (20), who showed increases in total muscarinic receptor density after 1 wk of denervation and decreases in total receptor density after 1 wk of diversion or diversion with denervation. While a trend for an increase in total receptor density was reported in the bladders induced to hypertrophy by paraffin injection, the increase was not significant (20), whereas we found a significant increase in total receptor density as a result of hypertrophy induced by outlet obstruction. This discrepancy may be the result of either the duration of hypertrophy or the method used to induce the hypertrophy.
In the DIV and DIV-DEN groups, despite decreased M3 receptor density (Fig. 4), the contractions are mediated by the M3 subtype (Table 1) and there is no change in agonist potency (Fig. 3). In other words there is no correlation between M3 receptor density and the potency of agonist or antagonist. The potency of carbachol is increased only in the DEN group (Fig. 3), which also has the largest increase in M2 receptor density (Fig. 4). The potency of carbachol is not increased in the BOO and MPG-DEC groups despite bladder hypertrophy and increased M2 receptor density. Thus the potency of carbachol for inducing contraction is not related to total receptor density or the density of the M2 or M3 subtype. This finding is similar to that reported by Nilvebrant et al. (20), who found that total receptor density does not correlate with supersensitivity in denervated, hypertrophied, diverted, or diverted and denervated bladders.
The significance of alterations in receptor density is not completely clear. However, the density of the M2 receptor subtype correlates with functional denervation. The greater the degree of functional denervation, the greater is the density of the M2 receptor subtype (Fig. 5A). No such correlation exists for the M3 subtype (Fig. 5B). However, all groups with bladder hypertrophy have an increase in M2 receptor density and low affinities for M3-selective antagonists, which suggests an M2-mediated component of contraction. The DIV and DIV-DEN bladders, which are not hypertrophied, have higher affinities for these M3-selective antagonists, suggesting that the M3 subtype mediates bladder contraction. Another report on the effect of BOO on muscarinic receptor-mediated bladder contraction found no differences in carbachol-mediated contraction (18). The differences in results reported by this group and our results may be due to the use of males as opposed to females or the duration of obstruction (4 wk as opposed to 3 days in our study).
Prejunctional autoreceptors have been identified on the nerves innervating the rat bladder (3, 26, 28). Activation of the M1 subtype increases acetylcholine release and contraction while activation of the M2 subtype reduces acetylcholine release and inhibits contraction. Somogyi and de Groat (27) have shown that the prejunctional facilitatory mechanism is upregulated after chronic spinal transection; furthermore, this facilitation appears to be primarily mediated by M3 receptors as opposed to M1 receptors in normal animals. Thus previous evidence exists for plasticity in the neural mechanism governing bladder contraction, and our results provide evidence for plasticity in the smooth muscle mechanism mediating bladder contraction.
The in vitro results reported in this manuscript, namely an M2
receptor-mediated component of contraction, is superficially in agreement with
in vivo experiments implicating the M2 receptor subtype in bladder
contraction. The amplitude of volume-induced bladder contractions in the
urethane-anesthetized rat is inhibited by subtype-selective muscarinic
antagonists with potencies that correlate most favorably with
pKi estimates of these compounds at human recombinant
M2 receptors (16).
In addition, the M2-and M4-selective antagonist AQ-RA
741, similar to the nonselective antagonist tolterodine, has a greater
selectivity for inhibition of bladder contraction than for inhibition of
salivation in the 
-chloralose-anesthetized cat, suggesting
M2 receptor involvement in bladder contraction
(15). Intravenous injection of
AF-DX116 (M2-selective antagonist) reduces contraction pressure but
not frequency or the duration of bladder contraction
(21). In these in vivo studies
of systemic administration of antagonists, the sites of action of the
antagonists are not known and inhibition of bladder contraction or salivation
could be due to additional effects on the central or the peripheral nervous
system and not strictly due to effects of the antagonist on the end organ
itself.
Intracerebroventricular injection of the M3-selective antagonist 4-DAMP inhibits both the amplitude and the duration of volume-induced bladder contractions, whereas AF-DX116 decreases contraction frequency while prolonging the duration of contraction (21). On the other hand, intracerebroventricular injection of darifenacin, an M3-selective antagonist, has no effect on voiding parameters in normal conscious rats, while intracerebroventricular injection of tolterodine, a nonsubtype-selective antagonist, decreases voiding pressure and increases bladder capacity (17). Thus evidence exists for the central nervous system control of bladder contraction by both M2 and M3 muscarinic receptor subtypes. Because the prejunctional facilitatory autoreceptors have been shown to change from M1 to predominantly M3 after spinal cord transection in the rat (27), it may also be possible that pathophysiological conditions could induce alterations in the central mechanisms governing micturition.
The question arises as to how M2 receptors directly mediate
smooth muscle contraction. M2 receptors are traditionally thought
to preferentially couple to PTX-sensitive G proteins such as the Gi
subfamily, resulting in inhibition of adenylyl cyclase, while M3
receptors preferentially couple to Gq and stimulation of
phosphoinositol hydrolysis leading to an increase in cytosolic calcium.
Pharmacological studies demonstrate that most smooth muscle contraction is
mediated by the M3 subtype
(7,
9). However, an
M2-mediated contractile response in bladder muscle can be
demonstrated after the majority of M3 receptors are inactivated in
an environment of increased intracellular cAMP such as during stimulation with
a 
-adrenergic agonist (5,
16). This pathway has been
proposed to mediate contraction indirectly, merely by blocking
-adrenergic agonist-induced relaxation via increased cAMP
(9). However, M2
receptors acting through Gi may also stimulate bladder contraction
directly via PKC activation as previously found in the cat lower esophageal
sphincter smooth muscle. A low degree of muscarinic stimulation and,
consequently, a low degree of calcium mobilization result in activation of
PKC, whereas PKC activation is inhibited at higher intracellular calcium
concentrations (24). Thus in
the face of normal calcium mobilization mediated by the M3 receptor
subtype, the signal transduction pathway mediated by the M2 subtype
may be inhibited. One hypothesis to explain the shift in muscarinic receptor
subtype mediating contraction from M3 to M2 in the
hypertrophied bladders is a deficit in calcium mobilization.
We have previously shown quantitative evidence for an interaction between the second messenger systems activated by the M2 and the M3 receptor subtypes in the denervated rat bladder (6). The simultaneous action of M2-selective and M3-selective antagonists induces a synergistic inhibition of contraction in denervated bladders, which indicates a facilitatory interaction of the two subtypes in inducing contraction. However, results in the normal rat bladder show no facilitatory interaction between M2 and M3 subtypes for inducing contraction. Only after either denervation or blocking the sarcoplasmic reticulum calcium pump with thapsigargin does this interaction become facilitatory. These two results provide further support for an interaction between subtypes mediating contraction as previously reported in the guinea pig colon where the M2 and M3 receptor subtypes are thought to interact in a facilitatory manner to mediate contraction (23). However, our results in the normal rat bladder do not support such a facilitatory interaction; actually, the opposite appears to occur, namely that the M3 pathway seems to inhibit the M2 pathway, possibly via calcium mobilization. It is possible that the interaction between subtypes is different in the guinea pig colon or that after 3 days of in vivo PTX treatment (23), the interaction between subtypes becomes altered.
Experimental pathologies that interfere with the normal functioning of the bladder induce a decrease in density of the M3 receptor subtype. Conditions that lead to hypertrophy induce an increase in density of the M2 receptor subtype and a shift in the mechanism of contraction such that the M2 subtype can be shown to at least partially mediate contraction.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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