BETHANECHOL ACTIVATES A POST-RECEPTOR NEGATIVE FEEDBACK MECHANISM IN RABBIT URINARY BLADDER SMOOTH MUSCLE

Vol. 169,252-257, Jan1998 Printed in U S A .

BETHANECHOL ACTIVATES A POST-RECEPTOR NEGATIVE FEEDBACK MECHANISM IN RABBIT URINARY BLADDER SMOOTH MUSCLE OFER Z. SHENFELD, CHARLES W.MORGAN

AND

PAUL H. RATZ*

From the Departments of Urology and Pharmacology, Eastern Virginia Medical School, Norfolk, Virginia

ABSTRACT

Purpose: Recent studies using vascular and gut smooth muscles indicate that contractile receptor agonists may activate post-receptor down-regulatory mechanisms causing a temporary reduction in the strength of subsequent contractions. Our data indicate a similar mechanism exists in detrusor smooth muscle of the urinary bladder. Materials and Methods: Each isolated strip of female rabbit detrusor was placed in a tissue bath, secured to an isometric force transducer, and length-adjusted until depolarization with 110 mM KC1 produced a maximum contraction (So).Subsequent contractions were normalized to So (S/S,)or to a first stimulus with 30 mM KC1 or caffeine (S/S1). Tissues were pretreated with the muscarinic receptor agonist, bethanechol (BE),then stimulated with KCl, caffeine, or Bay k 8644 to identify potential post-receptor down-regulation. Results: Contractions induced by 30 mM KCl had three phases labeled fast peak (FP), slow peak (SP)and steady-state (SS).In tissues exposed for 30 min. to a maximum BE concentration then washed for 5 min., the KC1-induced FP and SP, but not SS, responses were reduced by -40%. Smaller reductions in peak KC1-induced contractions occurred in tissues pretreated for a shorter duration or with a 100-fold lower BE concentration. This down-regulation induced by bethanechol pretreatment was reversible, lasting 1-2 h. Not only were KC1-induced contractions reduced by BE pretreatment, but also those produced by the intracellular Ca2+-mobilizer, caffeine, and the I.-type Ca2+ channel agonist, Bay k 8644. Conclusions: Pretreatment of isolated strips of rabbit detrusor with a muscarinic receptor agonist produced short-term down-regulation of KC1-induced peak contractions that may have involved inhibition of both influx of extracellular Ca" and release of intracellular Ca2+.Reductions in the degree of this novel modulatory response during disease conditions and aging could enhance contractile activity, possibly causing detrusor instability.

-

KEYWORDS: detrusor, heterologous desensitization, down-regulation Receptor stimulation not only causes smooth muscle contractions, but also may activate a negative feedback mechanism reducing the responsiveness to subsequent stimuli.' This well-documented modulatory mechanism is attribub able, in part, to homologous receptor desensitization, where receptor activation causes down-regulation of the receptor type that is activated.2 In the detrusor smooth muscle of the urinary bladder, for example, both muscarinicg and purinergie4 receptors undergo homologous desensitization. In addition, a second, intrinsically more complex form of down-regulation that can likewise limit the degree of responsiveness to contractile stimuli,termed heterologous desensitization, may also be activated by receptor stimulation.2 This negative feedback mechanism may involve receptor-induced down-regulation of signaling systems beyond the level of the receptor (posbreceptor down-regulation). Recent studies show that this negative feedback mechanism is welldeveloped in vascular and gut smooth muscles,610 and that it ma involve both a decrease in Ca2+influx and intracellular Ca release and a decrease in the sensitivity of contractile proteins to [Ca2+h.9.10Whether a similar mechanism modulates detrusor smooth muscle contractile activity remains to be determined. In patients with increased urgency and frequency of urination, includingurge incontinence,the bladder is thought to exhibit a lower capacity for urine storage and a heightened

sensitivity to distention. The cellular mechanisms responsible for this condition, known as irritable or hyperreflexic bladder, are not known. However, antimuscarinic compounds that effectively reduce destabilized detrusor contractions by acting at the neuromuscular junction indicate that a peripheral mechanism may be involved.11.12 If short-term, postreceptor down-regulation is a normal, physiological regulatory mechanism used by detrusor smooth muscle to limit the degree of tissue reactivity, then a decline in this negative feedback function with aging or under certain disease conditions could theoretically enhance spontaneous contractile activity or reactivity to neurogenic stimuli such as acetylcholine and ATP. For this reason, we determined whether postreceptor down-regulation exists in rabbit detrusor by examining the effect of muscarinic receptor activation on the ability of KC1, caffeine or Bay k 8644 to subsequently produce strong contractions.

z+

MATERIALS AND METHODS

Tissue preparation. Tissues were prepared as described previously for rabbit and pig arteries.6.13 Whole bladders from 18 mature female New Zealand white rabbits weighing approximately 2.5 kg. were removed immediately after the animals were killed by overdose with pentobarbital. Bladders were washed several times and stored for up to 48 h in cold ( 0 - 4 0 physiological salt solution (PSS),composition in m M NaCl, 140; KC1, 4.7; MgSO,, 1.2; CaCl,, 1.6 Na,HPO,, 1.2; morpholinopropanesulfonic acid, 2.0 (adjusted to pH 7.4 at either 0 or 37C, as appropriate); Na, ethylenediamine tet-

for publication July 14,1997. for. ~ p + k Department of Pharmacology, P.O. Box 1980, astam Vi-a Medical School,Norfolk VA 23501. 252

253

BETHANECHOL-INDUCED DOWN-REGULATION OF DETRUSOR CONTRACTIONS

raacetic acid (EDTA, to chelate trace heavy metals), 0.02;and dextrose, 5.6. High purity water (greater than 17MR distilled and deionized) was used throughout. Longitudinal muscle strips were cut from the wall of the detrusor body. Each muscle strip was incubated in aerated PSS at 37C in a water-jacketed tissue bath (Radnotti Glass Technology, Monrovia, CA) and secured by small clips t o a micrometer for length adjustments and an isometric force transducer (model 52,Harvard Apparatus, South Natick, MA) to measure muscle contraction. Contractions of isoZated detrusor strips. Isometric contractions were measured as described previously.6.13 Voltage signals were digitized (model DIO-DAS16, ComputerBoards, Mansfield, MA), visualized on a computer screen as force (gm.), and stored to a hard disk for later analyses. All data analyses were performed using customized computer programs written in compiled BASIC and an electronic spreadsheet (Quattro Pro, Borland International, Scotts Valley, CA). Contractile stress was measured as described previously.6.13 Tissues were equilibrated for 1h at -0.5 g m . passive force, then stretched to their optimum length for muscle contraction (Lo) using an abbreviated length-force determination.6.13-15 Values for tissue cross-sectional area and active stress (S; force per tissue cross-sectional area in N/m2) were calculated as described previously,13and the optimum stress for muscle contraction (So) produced by 110 mM KC1 at Lo was obtained for each muscle strip. To reduce tissue-to-tissue variability, subsequent contractions were reported as normalized to So (S/So) or as normalized to the response produced by a first stimulus (S/S,). In all studies using KC1, caffeine or Bay k 8644 as a stimulus, 0.1 pM atropine was added to prevent potential activation of muscarinic receptors resulting from depolarization of parasympathetic nerves. Statistics. Analysis of variance and the Student-NewmanKeuls test, or the t test, was used where appropriate to determine significance, and the Null hypothesis was rejected a t p <0.05.The population sample size (n value) refers to the number of animals, not the number of tissues.

1.2

l,o

l

.

l

.

.

.

I

.

.

.

.

.

l

.

.

.

.

.

I

.

.

.

.

.

I

.

.

A. Control

2 1.2 v) ,..",

I . . . . . , . . ' . . , ' . . ' . I ' . . . . I . .

B. Bethanechol (BE)

0.6 0.4

0.2 0.0 0

120

80

180

-

Control ARar BE

-0.2

1

184

106

188

190

192

194

196

Time (min) FIG. 1. Exam le of protocol used in strips of rabbit detrusor to determine whet1er pretreatment with muscarinic agonist, bethanechol (BE), temporaril reduces strength of subsequent 30 mM KClinduced contraction. %achtissue was stimulated with KCl 4 times (S 4,) over 4.5 h period. To determine effect of pretreatment on Kbl-inducedcontractile activity, no drug (A), 100 FM BE ( B ) ,or 40 mM KCl (not shown) was added for 30 min. before S,. Res nses to S3from (A) and (€0are shown expanded in ( C ) .Fast peak slow peak (SP)and steady-state (SS) responses are identified in CA) and (C). Summary data are 111 fig. 2.

(b),

RESULTS

Effect of bethanechol pretreatment on KCI-induced contractions. Contractions induced by 30 mM KC1 were triphasic (fig. 1, C , control). Upon addition of KC1, contractions developed rapidly, reaching a maximum within -6-23 sec. (fast peak; fig. 1, C , FP). This initial phasic response was followed by a second transient contraction peaking at -59-112 sec. (slow peak, fig. 1, C, SP) before leveling off to produce a sustained contraction (steady-state; fig. 1, C , SS) that was approximately 0.4-fold the FP response. Furthermore, this triphasic contractile behavior could be exactly reproduced a t least 4 times over a period of approximately 4.5 h (fig. 1, A; stimuli S,S,, and fig. 2, open bars, stimuli S2-S,). However, if tissues were pretreated for 30 min. with a maximum effecthen washed for tive concentration of bethanechol(100 5 min. (fig. 1,B, BE) and subsequently stimulated with KC1, FP (S3;fig. 1,B and left cross-hatched bar of fig. 2,A) and SP (s3; fig. 1, B and left cross-hatched bar of fig. 2,B ) responses were significantly reduced compared to control contractions (S3; fig. 1, A and open bars of figs. 2, A and 2, B). The KC1-induced SS response was not affected by prior muscarinic receptor activation (S3; figs. 1and 2,C ) . The reduction in KC1-induced peak contractile responses produced in tissues that were pretreated with bethanechol was not caused by prior tissue activation per se, because tissues that were stimulated for 30 min. with 40 mM KC1 rather than with bethanechol, and washed for 5 min., produced a strong triphasic contraction similar to that produced by control tissues when subsequently stimulated with 30 mM KCl (S3; right cross-hatched bars of fig. 2).

a),

Likewise, the reduction in peak contractile responses were not caused by a time-dependent decline in tissue viability because a fourth stimulation with 30 mM KC1 (S,) approximately 1 h after washout of the third stimulation (S,) produced FP and SP response that were not different than the control responses (S,;figs. 1and 2).Furthermore, this result indicates that the reduction in peak responses produced by bethanechol pretreatment was reversible, suggesting that the effect may represent a specific physiological downregulatory mechanism and not a non-specific depression of excitation-contraction coupling or contractile protein activation. Pretreatment with bethanechol for shorter durations. To determine whether a bethanechol stimulation duration of less than 30 min. would also reduce the ability of KC1 to subsequently produce strong peak contractile responses, tissues were pretreated for 5 or 15 min. with 100 p.M bethanechol, washed for 5 min., and stimulated with 30 mM KC1. Interestingly, SP contractions were reduced by nearly 40%by only 5 min. of bethanechol pretreatment, and FP contractions were reduced by approximately 30% by a 15 min. bethanechol pretreatment (fig. 3; 30 min. pretreatment WpOnse8 from fig. 2 are also included for a comparison). Pretreatment with a lower bethunechol concentration. TO determine whether bethanecholconcentratiom lese than 100

264

BETHANECHOL-INDUCEDDOWN-REGULATION OF DETRUSOR CONTRACTIONS

-A-

Fast Peak (FP)

+Slow Peak (SP)

BE W e e n S, 8 S, 629 KCI Between S, 8 S,

-0- Steady State (SS)

'-O

L t +_ r

I I

t 2

- B. Slow Peak (SP)

o*6 0.4

1

?

1

o-2 0.0

0 0

I

I

5

10

15

20

25

30

Bethanechol Duration (min)

I

2b

FIG.3. FP,SP and SS contractile responses to 30 mM KCI produced after tissues were pretreated with 100 pM BE for 5, 15,or 30 min. and washed for 5 min. to remove BE. For each tissue, responses produced by KC1 after washout of BE (S)were normalized to those produced before BE addition (S1). Data shown at time = 0 are from control tissues that were not pretreated with BE. n = 3-4; * p <0.05 compared to control.

C. Steady-State (SS)

s*

s3

s4

1 .o

Contraction a

FIG.2. Effect of pretreatment for 30 min. with 100 bethanechol on KC1-induced contractions. Shown are average contractile responses to three consecutive stimulations with 30 mM KCI (Ss4) normalized to firstresponse (S). Detrusor strips were exposed to 100 BE (lefbhatched bars) or 40 mM KC1 (right-hatched bars) for 30 min.. then washed for 5 min. before S ,. Duration between Sz & S, was -1.5 h, andbetweenS,&S, was -1 h(seefig. 1). n = 3-4; * p <0.05 compared to control.

0.5

pM could also reduce the ability of KC1 to subsequently produce strong peak contractile responses, tissues were pretreated for 30 min. with 1 @ bethanechol, washed for 5 0.0 min., and stimulated with 30 mM KCl. This bethanechol concentration produced approximately a half-maximal contraction in rabbit detrusor. As with 100 @, 1@l bethanechol pretreatment also reduced both F" and SP contractile responses, but not the SS response (fig. 4). The degree of FIG.4. FP,SP and SS contractile responses produced by second inhibition was less than that produced by pretreatment with KCI stimulus (S)in control tissues ( 0 en bars) and in tissues re 100 pM PE (please compare fig. 4 with Ssof fig. 2). treated for 30 min. with 1 pM BE an! washed for 5 min. (hatcted Rewrsibility. To determine the degree of reversibility of bars). Res nses were normalized to those produced by first KCI the bethanechol-induced inhibition of KC1-induced peak con- stimulus (&. n = 3; * p c0.05 compared to control. tractile activity, tissues pretreated for 30 min. with 100 bethanechol were washed for between 5 and 70 min. before they were exposed to KCl. The strength of KC1-induced con- period of 60-70 min.permitted KCl to produce stronger peak tractions were proportional to the duration between washout responses of approximately 0.8-fold S,. Based on the apparof bethanechol and addition of KCl (fig. 5). That is, aRer 5 ently linear relationship between the "resting" duration and min. had elapsed between washout of bethanechol and addi- the strength of KC1-induced peak contractile response, extion of KCl, peak contractions were only approximately 0.45- trapolation of the data to 1-fold S, (i.e., the level of contracfold the first KC1-induced contraction (Sl). A longer "resting" tion produced by the first stimulus with KC1; dotted line of

ss SP Responses to 30 mM KCI FP

1-1

1.2 i

BETHANECHOL-INDUCED DOWN-REGULATION OF DETRUSOR CONTRACTIONS '

l

'

l

'

l

'

l

'

l

'

l

I

1

After 100 pM BE

Slow Peak (SP)

1.o

n

255

T

1

0.8

0.6 -0

0.4 -

I

.

I

,

I

,

I

,

I

.

,

Duration After BE Washout (min) FIG.5. Reversibility of down-regulation.Tissues pretreated for 30 min. with 100 BE were washed to cause complete relaxation, then contractecf%th 30 mM KC1 5 to 70 min. after BE washout. Extrapolation of data to 1 (dashed line) suggests that responses may return to control levels (S,)within 2 h. n = 3.

fig. 5) suggests that complete restoration of peak contractile activity may require approximately 2 h. Apparently, the time it took to completely restore peak contractile responses was reduced when a stimulus occurred shortly after washout of bethanechol. This conclusion is based on the observation that KC1-induced FP and SP responses produced approximately 75 min. after bethanechol pretreatment were equivalent to control responses in tissues that had a KC1 stimulus (S,) interposed between bethanechol washout and the KC1 stimulus given 75 min. later (please compare S, responses of figs. 1 and 2 with responses shown in fig. 5). Effect of bethanechol pretreatment on caffeine- and Bay k 8644-induced contractions. Contractions produced by KC1 are primarily caused by depolarization-induced activation of Ltype Ca2+ channels and subsequent increases in the rate of Ca2+ influx.16 However, the increase in [Ca2+li brought about by enhanced Ca2+ influx may itself cause increased release of intracellular Ca2+ stored in the sarcoplasmic reticulum (Ca2+-induced Ca2+-release, or CICR).16 It is therefore possible that the reduction in KC1-induced contractions produced by bethanechol pretreatment occurred because of reduced L-type Ca2+ channel activity or reduced CICR. To test this hypothesis, rather than contracting with KC1 after pretreating with bethanechol, tissues were contracted with either Bay k 8644 (0.1 an L-type Ca2+ channel activator, or caffeine (20 mM), an activator of CICR. Caffeine produced rapid, transient contractions lasting no more than 10-20 seconds. Three reproducible peak contractile responses could be produced over an approximately 3 h period when tissues were exposed to caffeine for a very short duration (less than 20 sec). When normalized to the peak response of the first caffeine stimulus (Sl), the peak response to a second stimulus produced 5 min. after pretreating tissues for 30 min. with 100 pM bethanechol was approximately 0.5-fold that produced by control tissues that were not pretreated (S2;fig. 6). Furthermore, the peak response to a third caffeine stimulus given approximately 1 h after washout of caffeine from the second stimulus was as strong as the fig. 6). control response (S3; Contractile responses to the Ca2+-channel activator, Bay k

a),

Responses to 20 mM Caffeine FIG. 6. Effect of bethanechol retreatment on contractions produced by intracellularCa2'-mobiyizing agent, caffeine. Tissues were treated three times with 20 mM caffeine and second (S ) and third (S ) peak responses were normalized to first Belore second &eine treatment, tissues were pretreated for 30 min. with 100 ph4 BE (hatched bars) or no drug (control; open bars) and washed for 5 min. to cause complete relaxation. Duration between S, and S, was -1 h. n = 4; * p <0.05.

8644, were not completely reproducible when given consecutively, as were KC1- and caffeine-induced contractions. Furthermore, responses were often highly rhythmic, displaying neither clear peak nor steady-state contractions within a 10 min. period (fig. 7, A). For this reason, to quantify contractile responses to Bay k 8644,measurements were made of the area under the curve (AUC) for a contraction-duration of 5 min. Tissues stimulated with 0.1 pM Bay k 8644 that had been pretreated for 15 or 30 min. with 100 pM bethanechol and washed for 5 min. produced contractile responses that were less than 0.4-fold that produced by control tissues that had not been pretreated (fig. 7). DISCUSSION

To assess whether receptor activation produces postreceptor down-regulation of contractions, smooth muscle must be contracted by a mechanism that acts similarly to receptor activation, but that bypasses receptors. KC1 provides such a stimulus because hi h levels depolarize the membrane, causing increased [Ca Ii, myosin light chain phosphorylation and contraction.16 Rf sults from the present study indicate that post-receptor down-regulation can be activated in detrusor smooth muscle by pretreatment with the muscarinic receptor agonist, bethanechol. As seen in vascular smooth musclef the degree of reduction in the ability of detruaor strips to produce strong KC1induced peak contractions appeared to be dependent on both the duration of pretreatment with, and concentration of, the contractile receptor agonist. Furthermore, as seen in vascular smooth muscle,6 the down-regulation was completely reversible (see fig. 5 and S, in figs. 1& 2). However, the rate at which these responses returned towards the control level appeared to depend on whether tissues were contracted with KC1 only once (asin fig. 5)or twice (as in figs. 1and 2) aRer the pretreatment period. Interestingly, unlike that seen in vascular smooth muscle,6 postreceptor down-regulation in

Q+

BETHANECHOGINDUCED DOWN-REGULATION OF DETRUSOR CONTRACTIONS

256

3

-0.2 0

2

6

4

8 1 0 1 2

Time (min) 100 0 X

o v) W

*3

60

eZm After 15' BE After 3 0 BE

40

20

a . Responses to 0.1 pM Bay k 8644 FIG.7. Contractions (A, t i d B , area under the curve (AUC)) produced by 0.1 pM of the? type CaZ' channel activator, Bay k 8644. Detrusor stri were pretreated for 15 or 30 min. with 100 pM BE (A, after B E B,ratched bars) or no drug@, control; B, open bar), washed for 5 min. to cause complete relaxation, then contracted mth Bay k 8644. n = 4 6 ; * p (0.05 compared to control.

'

vascular smooth muscle, either Ca2+ release from intracellular stores was temporarily inhibited, or intracellular Ca2+ stores were not completely replenished following aadrenergic receptor activation. The latter mechanism supports the general model put forth by van Breemen and colleagues, in which it is proposed that the intracellular Ca2+ store of smooth muscle serves as a superficial buffer barrier for Ca2+ entering the cell, limiting the degree of contractile protein activation.21 Moreover, recent studies have reported that the intracellular Ca2+ store in bladder smooth muscle also behaves as a superficial buffer barrier, diverting some of the Ca2+ entering the cell away from the cytosolic compartment.22 Detrusor contains both m2 and m3 subtypes of muscarinic receptors, and in rabbit bladder the ratio of these subtypes is 3:1.23 Activation of the m3 receptor subtype appears to correlate with detrusor contractior1,2~but the role for the m2 receptor subtype in this tissue remains to be determined. It is tempting to speculate that m2 receptors play a role in the down-regulation seen in the present study. However, as suggested by Wang et al.,23 it is likely that m2 receptors are located on presynaptic nerve terminals and modulate release of acetylcholine. Additional data are required to identify which muscarinic receptor subtype activates subsequent post-receptor down-regulation, and to determine how muscariNc receptor activation produces this modulatory response. Detrusor motor nerve activation results in a rapid increase in pressure that subsides quickly followed by a weaker but sustained phase of elevated pressure.24 The early response is attributed to neuronal release of both ATP and acetylcholine, resulting in activation of detrusor smooth muscle purinergic and muscarinic receptors, respectively.24-27 Interestingly, contractions produced by purinergic receptor activation are entirely dependent on influx of extracellular Ca2+ through Gtype channels.28 Furthermore, they are more sensitive to the dihydropyridine Ca2+ channel agonist and antagonist, Bay k 8644 and nifedipine, respectively, than are steadystate contractions produced by muscarinic activation.29 Conversely, the rapid phase of contraction due to muscarinic receptor activation appears to be more dependent on mobilization of intracellular Ca2+than on Ca2+influx.26,27Thus, it is possible that by reducing both intracellular Ca2+mobilization and a component of Ca2+ influx that is highly sensitive to the effects of dihydropyridines, post-receptor desensitization may temporarily reduce the ability of motor nerve stimulation to initiate micturition. If such a negative feedback mechanism is a normal component of detrusor reactivity to contractile stimuli, then it is theoretically possible that alterations in the degree of feedback modulation may participate in pathologies associated with altered detrusor contractile activity.

detrusor involved a temporary reduction in the magnitude of KC1-induced initial peak, but not steady-state, contractile activity. However, as in both gut and vascular smooth muscle,s-'o this negative feedback mechanism appeared to involve reductions in mobilization of Ca2+. Taken together, these data support the hypothesis that down-regulation of the rapid, peak components of KC1induced detrusor contractions may reflect a physiological negative feedback mechanism temporarily limiting the degree of muscle activation for a short duration following muscarinic receptor stimulation. Moreover, studies with the selective agents, caffeine and Bay k 8644, indicate that the CONCLUSION down-regulation involved decreases in Ca" mobilization. Pretreatment of isolated rabbit detrusor for between 5 and For example, bethanechol pretreatment greatly reduced the ability of the selective Gtype Ca2+ channel activator, Bay k 30 min. with submaximum and maximum concentrations of 8644," to produce contractions, suggesting that down-re- the muscarinic receptor agonist, bethanechol, produced temN a t i o n involved reduced voltage-dependent Ca2 channel porary down-regulation of KC1-induced peak contractions. activity. Voltage-dependent Caz+ channel activity in blad- Steady-state contractions were unaffected by bethanechol der18 and intestinal19 smooth muscle cells has been shown to pretreatment. Reductions in KC1-induced peak contractile be reduced by muscarinic receptor activation. Interestingly, responses may have involved inhibition of both influx of in the latter case,19 inhibition of Ca2+ channel current re- extracellular Ca2+ and release of intracellular Ca2 . Almained for some time after removal of the muscarinic recep- though purely speculative, a reduction in the effectiveness of this novel negative feedback mechanism during disease contor stimulus. Pretreatment of rabbit detrusor with bethanechol also re- ditions or with aging would enhance contractile activity that duced the ability of caffeine, a mobilizer of intracellular could theoretically lead to detrusor instability. Caa+,18*20to cause a contraction. In the rabbit femoral arREFERENCES tery, pretreatment with the a-adrenergic receptor agonist, phenylephrine, also reduces the ability of caffeine to subse1. Furchgott, R. F. and Bhadrakom, S.: Reactions of strips of rabbit quently produce strong contractions and concomitantly to aorta to epinephrine, sodium nitrate and other drugs. J. Pharproduce large increases in [Ca2+Ii.loIt was proposed that, in macol. Exp. Ther., 108 129,1953. +

+

BETHANECHOL-INDUCEDDOWN-REGULATION OF DETRUSOR CONTRACTIONS

2. Benovic, J. L., Bouvier, M., Caron, M. G. and Lefiowitz, R. J.: Regulation of adenylyl cyclase-coupled P-adrenergic receptors. Ann. Rev. Cell Biol., 4:405,1988. 3. Marsh, K. A.,Harriss, D. R. and Hill, S. J.: Desensitization of muscarinic receptor-coupled inositol phospholipid hydrolysis in human detrusor cultured smooth muscle cells. J. Urol., 155: 1439,1996. 4. Kasakov, L.and Burnstock, G.: The use of the slowly degradable analog, a$-methylene ATP, to produce desensitization of the P,-purinoceptor: effect of non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder. Eur. J. Pharmacol., 8 6 291, 1983. 5. Declerck, I., Himpens, B., Droogmans, G. and Casteels, R.: The a,-agonist phenylephrine inhibits voltage-gated Ca2+channels in vascular smooth muscle cells of rabbit ear artery. Pflugers Arch., 417: 117,1990. 6. Ratz, P. H.: Receptor activation induces short-term modulation of arterial contractions: memory in vascular smooth muscle. Am. J. Physiol., 269 C417,1995. 7. Mitsui, M. and Karaki, H.: Dual effects of carbachol on cytosolic Ca2+ and contraction in intestinal smooth muscle. Am. J. Physiol., 258 C787, 1990. 8. Hishinuma, S., Matsumoto, Y., Uchida, M. K. and Kurokawa, M.: Novel regulation of muscarinic receptors and their coupling with G proteins in smooth muscle: transient resensitization during desensitization process. Br. J. Pharmacol., 109: 330,1993. 9. Ratz, P. H., Lattanzio, F. A., Jr. and Salomonsky,P.-M.: Memory of arterial receptor activation involves reduced [Ca2'li and desensitization of cross bridges to [Ca2+li.Am. J. Physiol., 269. C1402, 1995. 10. Ratz, P. H.,Salomonsky, P.-M. and Lattanzio, F. A., Jr.: Memory of previous receptor activation induces a delay in [Ca2'li mobilization and decreases the Ca2'-sensitivity of arterial contractions. J. Vasc. Res., 847: 489,1996. 11. Coolsaet, B. L.R. A., Van Duyl, W. A, Van 0s-Bossagh, P. and De Bakker, H. V.: New concepts in relation to urge and detrusor activity. Neurourol. Urodyn., 1 2 463,1993. 12. Ferguson, D. and Christopher, N.: Urinary bladder function and drug development. Trends Pharmacol. Sci., 17: 161,1996. 13. Ratz, P. H. and Murphy, R.A.: Contributions of intracellular and extracellular Ca2+pools to activation of myosin phosphorylation and stress in the swine carotid media. Circ. Res., 60:410, 1987. 14. Herlihy, J. T. and Murphy, R. A.: Length-tension relationship of smooth muscle of the hog carotid artery. Circ. Res., 33: 257,

257

variations in cell length in longitudinal smooth muscle from rabbit urinary bladder. A d a Physiol. Scand., 97: 1, 1976. 16. Himpens, B., Missiaen, L. and Casteels, R.: Ca" homeostasis in vascular smooth muscle. J. Vasc. Res., 3 2 207,1995. 17. Schramm, M., Thomas, T., Towart, R. and Franckowiak, G.: Novel dihydropyridines with positive inotropic action through activation of Ca2+ channels. Nature, 303 535,1983. 18. Yoshino, M. and Yabu, H.: Muscarinic suppression of Ca2+ current in smooth muscle cells of the guinea-pig urinary bladder. Exp. Physiol., 80: 575, 1995. 19. Unno, T., Komori, S. and Ohashi, H.: Inhibitory effect of musearinic receptor activation on Ca2+ channel current in smooth muscle cells of guinea-pig ileum. J. Physiol., 4&4:567,1995. 20. Endo, M., Yagi, S., Iino, M.: Tension-pCa relation and sarwplasmic reticulum responses in chemically skinned smooth muscle fibers.Fed. Proc., 41: 2242,1982. 21. van Breemen, C., Chen, Q. and Laher, I.: Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum. Trends Pharmacol. Sci., 16 98,1995. 22. Yoshikawa, A, van Breemen, C. and Isenberg, G.: Buffering of plasmalemmal Ca2+ current by sarcoplasmic reticulum of guinea pig urinary bladder myacytes. Am. J. Physiol., 271: C833,1996. 23. Wang, P., Luthin, G. R. and Ruggieri, M. R.: Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J. Pharmacol. Exper. Ther., 273 959,1995. 24. Levin, R. M., Ruggieri, M. R. and Wein, A. J.: Functional effects of the purinergic innervation of the rabbit urinary bladder. J. Pharmacol. Exp. Ther., 238:452,1986. 25. Chancellor, M. B., Kaplan, S. A. and Blaivas, J. G.: The cholinergic and purinergic components of detrusor contractility in a whole rabbit bladder model. J. Urol., 148: 906,1992. 26. Iacovou, J. H.,Hill, S. J. and Birmingham, A. T.: Agonistinduced contractions and accumulation of inositol phosphates in the guinea-pig detrusor: evidence that muscarinic and purinergic receptors raise intracellular calcium by different mechanisms. J. Urol., 144: 775,1990. 27. Zhao, Y., Wein, A. J. and Levin, R. M.: Role of calcium in mediating the biphasic contraction of the rabbit urinary bladder. Gen. Pharmacol., 24: 727,1993. 28. Katsuragi, T., Usune, S. and Furukawa, T.: Antagonism by nifedipine of contraction and Ca2+-influx evoked by ATP in guinea-pig urinary bladder. Br. J . Pharmacol., 100:370,1990. 29. Bo, X. N. and Burnstock, G.: The effects of Bay K 8644 and nifedipine on the responses of rat urinary bladder to electrical field stimulation, beta, gamma-methylene ATP and acetylcho1972 __ ._. line. Br. J. Pharmacol.. 101: 494. 1990. 15. Uvelius, B.:Isometric and isotonic length-tension relations and ~