The contribution of intracellular Ca²⁺release to contraction in human bladder smooth muscle

Abstract

1. The importance of Ca²⁺ release from the sarcoplasmic reticulum (SR) in excitation contraction (EC) coupling in human detrusor muscle remains controversial. In this paper the contribution of Ca²⁺ release to agonist induced contraction is assessed.

2. Dose response curves to carbachol (0.01–10 μM) were constructed before and after exposure to 200 nM Thapsigargin (Tg). Tg pre-treatment reduced the force of contraction at all agonist concentrations however, the reduction was dose dependent. At 0.1 μM the contractions were reduced to 14.5±7% (mean±s.e.mean) of controls (n=8) while at 10 μM the contractions were only reduced to 92±3% of controls (n=10).

3. The role of external Ca²⁺ was examined by measuring the magnitude of contraction to low and high doses of agonist in the presence and absence of external Ca²⁺. With (0.1–0.3 μM) carbachol the contractions in nominally Ca²⁺ free media were 4±4% of controls (n=7) whilst with (1–10 μM) carbachol the contractions were 36±8% of controls (n=7) suggesting that at low agonist concentrations the release of Ca²⁺ has a requirement for external Ca²⁺.

4. Pre-treatment of muscle strips with the Ca²⁺ channel blocking agent diltiazem reduced the contractile responses to carbachol. Contractions induced by 0.1 μM were reduced to 29±11% (P<0.05) of controls while those activated by 10 μM were reduced to 86±6% (P=0.1) of controls (n=4) suggesting the Ca²⁺ influx needed to activate internal store release at low agonist stimulation is through L-type Ca²⁺ channels.

5. These observations confirm the importance of thapsigargin sensitive intracellular Ca²⁺ store release in the activation of contraction of detrusor smooth muscle and suggest the overall contribution of this store depends upon the magnitude of the agonist stimulation.

Introduction

For many smooth muscle cell types, including detrusor smooth muscle, the cellular components involved in excitation contraction (EC) coupling have been identified and described. Information is available on the surface membrane receptors, voltage operated Ca²⁺ channels, receptor-linked phospholipase C, inositol trisphophate (IP₃) production, IP₃, Ca²⁺ activated channels on the sarcoplasmic reticulum, calmodulin and activation of the contractile proteins (Andersson, 1995; Chambers et al., 1996; Fry & Wu, 1997; Iacovou et al., 1990; Mostwin, 1985). The key event in the initiation of a contraction is the rise in cytoplasmic Ca²⁺. Two sources of Ca²⁺ have been identified which could contribute to this change; an influx of Ca²⁺ across the surface membrane and a release of Ca²⁺ stored within the sarcoplasmic reticulum (SR). The influx could involve at least three types of ion channel; voltage gated Ca²⁺ channels, receptor operated channels (Andersson, 1995), and Ca²⁺ release activated Ca²⁺ channels (Berridge, 1995). Two molecularly distinct intracellular Ca²⁺ release mechanisms have been identified in smooth muscle; an inositol trisphosphate (IP₃) and a Ca²⁺ and ryanodine sensitive, Ca²⁺ induced Ca²⁺ release (RyR-CICR) process (Somlyo & Somlyo, 1994). With respect to human detrusor the relative importance of the contribution of the different Ca²⁺ influx mechanisms and the release of Ca²⁺ from the IP₃ and RyR-CICR dependent release processes on the SR remains controversial. This paper describes experiments to identify the contribution of Ca²⁺ influx mechanisms and the release of intracellular Ca²⁺ release to carbachol induced contractions.

In the normal human bladder the major neurotransmitter is acetylcholine (ACh). However, ATP and numerous neuropeptides are also effective in initiating detrusor contractions (Andersson, 1995; Hasan & Neal, 1995). The current concept is that several neurotransmitters are co-released from the nerve terminals to initiate contraction (Burnstock, 1985). ACh activated contractions do not appear to be the result of membrane depolarization, action potential generation and Ca²⁺ influx (Brading & Inoue, 1991; Fry & Wu, 1997). Rather, the membrane hyperpolarizes, there is a Ca²⁺ influx via non selective cation channels and the release of intracellular Ca²⁺ release via an IP₃ dependent mechanism. On the other hand ATP can depolarize the detrusor smooth muscle and initiate Ca²⁺ dependent action potentials (Fry & Wu, 1997; Inoue & Brading, 1990, 1991). Such complex and varied effects of different neurotransmitters will add to the complexity of EC coupling in the human detrusor in vitro.

In order to assess the contribution of intracellular Ca²⁺ release to acetylcholine induced contraction one approach is to remove the contribution of Ca²⁺ stores. Thapsigargin (Tg), at low doses, is known to be a potent irreversible inhibitor of the sarcoplasmic reticulum Ca²⁺ pump (Thastrup et al., 1994). Concern has been raised about the specificity of Tg when micromolar concentrations are used as at these concentrations it may also block the L-type Ca²⁺ channels, (Buryi et al., 1995; Shmigol et al., 1995). However at nanomolar concentrations whilst inhibition of Ca²⁺ uptake into the SR can be demonstrated there does not appear to be an affect on the L-type Ca2+ channel in smooth muscle cells (Buryi et al., 1995). In the intact cell, inhibition of the pump allows Ca²⁺ to leak from the SR often resulting in a rise in cytosolic Ca²⁺ with an associated contraction. With prolonged exposure to Tg the Ca²⁺ content of the SR is reduced and consequently the effectiveness of the SR to contribute Ca²⁺ during EC coupling is diminished. In many and varied smooth muscle cell types Tg has been reported to decrease the magnitude of agonist induced contractions (Bian et al., 1991; Mikkelson et al., 1988; Neusser et al., 1993). This approach was used in the experiments described in this paper. The data confirm the importance of intracellular Ca²⁺ release in the activation of contraction of detrusor smooth muscle but reveal a level of complexity not previously reported.

Methods

Isolation of muscle strips

Detrusor muscle biopsies were obtained from patients during open and endoscopic bladder surgery with prior informed consent and with ethical committee approval from Newcastle Area Health Authority. Biopsy specimens were obtained mainly from the posterior and postero-lateral bladder walls during endoscopic surgery and the bladder dome following open surgical approach. In the present study no differences were detected between tissue sample obtained from the different anotomical locations or when the samples were obtained by endoscopy or open bladder surgery. No biopsies were taken from the bladder neck or the trigone area. Samples were collected in sterile RPMI 1640 medium (Life Technologies Ltd, Renfrewshire, U.K.), containing penicillin (100 μg ml⁻¹) and streptomycin (100 g ml⁻¹) and maintained at 4°C during transport from theatre. The detrusor was isolated by careful excision of the mucosal and serosal layers. Muscle strips approximately 5 mm in length and 1 mm in diameter were prepared. Experiments were carried out at 35–37°C in an organ bath containing oxygenated (95% O₂ and 5% CO₂) Tyrodes solution (mM): NaCl 120, NaHCO₃ 24, Glucose 6.1, Na Pyruvate 5, KCl 4, CaCl₂ 1.8, MgCl₂ 1 and NaH₂PO₄ 0.4 (pH 7.36), supplied by a continuously fed irrigation system (5–7 ml per min). The force generated by the muscle strips was recorded on a computer based data acquisition system (Sensonor and Newcastle Photometric Systems). In all experiments the strips were pre-tensioned to about 11 mN and then allowed to equilibrate for 60–90 min.

Solutions

Carbachol (Sigma) was made up as a 1 mM stock solution in distilled water and further diluted in the experimental solutions to the desired concentration at the time of the experiment. Thapsigargin (Sigma) was dissolved in DMSO and frozen in 2 mM aliquots and stored at −20°C. At the time of the experiment this was thawed and added to the experimental solutions to give a concentration of 200 nM. This concentration of thapsigargin was chosen based on a preliminary series of experiments to determine the effectiveness of low doses of thapsigargin to inhibit the uptake of ⁴⁵Ca²⁺ into the SR. Saponin permeabilized detrusor smooth muscle cells maintained in vitro were used as previously described (Chambers et al., 1996, 1999). Tg concentrations between 3–200 nM progressively inhibited the uptake of ⁴⁵Ca²⁺, the inhibition reaching a plateau at 200 nM which represented an 85% inhibition of total uptake. Higher concentration had no effect on this residual uptake which may represent non-specific binding of residual accumulation into organelles. On the assumption that cultured human bladder cells will behave as cells in intact tissue this concentration of Tg was chosen for all subsequent experiments. Nominally Ca²⁺ free solutions were made by excluding Ca²⁺ and adding the Ca²⁺ chelator EGTA (Sigma), (2 mM). Diltiazem (Sigma), an L-type Ca²⁺ channel antagonist was dissolved in 1 ml of DMSO and then made up to 10 ml of 10 mM stock with distilled water before further dilution in the experimental solutions to give a final concentration of 10 μM.

Statistical analysis

Mean data±s.e.mean are shown. Significance was tested using Student's t-test. P values <0.05 were considered significant. Where stated n values represent the number of subjects.

Results

The first series of experiments was done to determine the effect of removal of intracellular Ca²⁺ release on agonist (carbachol) induced contractions. In each experiment contractions were activated by brief exposure to increasing concentrations of agonist (0.01–10 μM; Figure 1A). Thapsigargin (Tg) was then added to the superfusion medium. In eight out of ten experiments Tg (200 nM) did not cause a contraction (see also Figure 2). This may be interpreted to suggest that, despite Ca²⁺ pump inhibition, the leak of Ca²⁺ from the SR is low and insufficient to raise cytosolic Ca²⁺ enough to initiate a contraction. In order to be certain that the internal stores were depleted of Ca²⁺ the muscle strips were exposed to a maximal dose of agonist. This will release stored Ca²⁺ but in the presence of Tg the stores will not refill. The strips were then re-exposed to the same range of concentrations of agonist to initiate contraction (Figure 1A). Tg pre-treatment reduced the amplitude of contraction at both high and low carbachol concentrations. Figure 1B shows the accumulated data from a total of ten experiments from different subjects illustrating the force produced before and after Tg treatment. Over the entire agonist concentration range Tg reduced the force of contraction supporting the idea that intracellular Ca²⁺ release contributes to contraction (P<0.05 for all concentrations).

Figure 1.

The effect of thapsigargin (Tg; 200 nM) on carbachol induced contractions of human detrusor smooth muscle. (A) Illustrates original data from a single experiment. Ordinate shows force produced by the muscle strip (mN) and the abscissa, time in s. Where indicated carbachol, was added to the superfusing solution. (The concentrations at points 1, 2, 3, 4 and 5 were 0.1, 0.3, 1, 3 and 10 μM respectively). Tg was added where indicated and subsequent exposure to carbachol carried out in the presence of Tg. All experiments were done at 35–37°C. (B) Shows combined data from ten separate experiments. The dose response curve to carbachol is shown for each strip before and after exposure to Tg. Mean values are shown±s.e. mean.

Figure 2.

The effects of thapsigargin on resting tension in isolated strips of human detrusor smooth muscle. (A) Shows one of three recordings in which a contraction was observed in response to Tg (200 nM). (B) Illustrates one of 33 recordings in which no overt contraction was seen. All experiments were carried out in gassed Tyrodes solution pH 7.36. Temperature 35–37°C.

Two series of control experiments were done in which either repetitive doses of carbachol were given over the entire dose range or two successive dose response curves were determined. There was no difference seen between the successive sets of responses in either of these series of control experiments (data not shown). Therefore the effects of Tg were not influenced by run down or alterations to the integrity of the preparations.

In other smooth muscle preparations exposure to Tg results in a transient contraction as a consequence of SR pump inhibition and the leakage of Ca²⁺ from the SR. In a total of 36 experiments on isolated human detrusor smooth muscle an overt contraction was only seen in three strips. Figure 2A illustrates one such contraction. In all of the other muscle strips no contraction was seen (Figure 2B).

There is a clear effect of Tg on subsequent agonist evoked contractions (Figure 1). Therefore, these data suggest that either the leakage of Ca²⁺ from the SR in human detrusor must be low and that the Ca²⁺ homeostatic mechanisms are sufficient to cope with an increased cytoplasmic Ca²⁺ load or that any leaked Ca²⁺ is successfully buffered away from the myofilaments.

Examination of Figure 1 suggests that Tg treatment has a relatively greater effect on small contractions compared to that on large contractions. This is illustrated directly in Figure 3A where the amplitude of the contraction in the presence of Tg is expressed as a percentage of the contraction in the absence of Tg and plotted against the agonist concentration. With low doses of carbachol, Tg treatment causes a near complete abolition of the contraction. With progressively greater doses of agonist the effect of Tg becomes less so that at the highest agonist concentrations used there is only an 8% reduction in the force of contraction. These data can be re-plotted to illustrate the relationship between the amplitude of contraction and the effectiveness of Tg. Figure 3B shows this relationship. It is clear that for small contractions there is almost a total dependence on intracellular Ca²⁺ release to activate a contraction. For large contractions there is less reliance on store release. These data can be interpreted to suggest that detrusor smooth muscle relies predominantly on intracellular Ca²⁺ release to activate small contractions but less so for large contractions. Thus, for large contractions a Ca²⁺ influx must contribute nearly all the Ca²⁺ needed to activate the contractile apparatus.

Figure 3.

The ratio of amplitudes of contractions in the absence and presence of Tg, expressed as percentages, are plotted against (A) the carbachol concentration and (B) the amplitude of the control contraction in the absence of Tg. Temperature 35–37°C. Mean values are shown±s.e. mean.

If the conclusions drawn from the above experiments are true, then, contractions activated by low doses of agonist should be of similar amplitude in the presence and absence of extracellular Ca²⁺. Figure 4 illustrates an experiment designed to examine this possibility. The original records in Figure 4A show first a contraction to 0.3 μM carbachol in the presence of external Ca²⁺ followed by a wash in nominally Ca²⁺ free medium and exposure to the same carbachol concentration. In the absence of Ca²⁺ there was no contraction. This was not what was expected from the previous analysis on the effects of Tg. On exposure to high agonist, 10 μM carbachol, a contraction was recorded in both Ca²⁺ containing and Ca²⁺ free solutions. Thus, the absence of any response at the low concentration cannot be a consequence of the stores being empty since they can be accessed by the higher concentration. The combined data from seven experiments are shown in Figure 4B.

Figure 4.

The amplitude of agonist induced contractions in the presence and absence of extracellular Ca²⁺. (A) Shows original data from a single experiment. Ordinate shows force produced by the muscle strip (mN) and the abscissa, time in s. Where indicated carbachol, at (1), 0.3 μM and (2), 10 μM was added to the superfusing solution. The superfusion solution was changed to a nominally Ca²⁺ free solution where indicated. (B) Illustrates data accumulated from seven separate experiments. Contractions induced by low (0.1–0.3 μM) moderate (1 μM) and high (10 μM) doses of carbachol, in the presence (open bars) and absence (filled bars) of extracellular Ca²⁺ are shown. Values are means±s.e.mean and all experiments were done at 35–37°C.

The response to 10 μM carbachol in nominally Ca²⁺ free was 38±11% (n=4) of the contraction in the presence of external Ca²⁺. This appears to indicate that the stores contribute a significant amount of Ca²⁺ to the contraction activated by 10 μM carbachol. This conclusion is in contrast to that derived from the Tg experiments where, at this agonist concentration, only 8% of the Ca²⁺ used for contraction is derived from the stores. One possible interpretation of these data is that the influx activated at high agonist concentrations reduced the amount of Ca²⁺ released from the store. This is in keeping with the published data on the effects of Ca²⁺ in IP₃ induced Ca²⁺ release (Bezprozvanny et al., 1991; Chambers et al.,1996; Hirose et al., 1998; Iino & Endo 1992; Missiaen et al., 1994).

The data in Figure 4 demonstrate that extracellular Ca²⁺ is important for the initiation of the agonist induced release from the Tg sensitive internal stores. This could suggest that a Ca²⁺ influx is essential during agonist stimulation in order to trigger intracellular release. Such an influx could occur via voltage operated Ca²⁺ channels or receptor operated Ca²⁺ channels or both. An insight into which type of channel might be involved was obtained using diltiazem, a blocker of voltage operated Ca²⁺ channels (Figure 5). Contractions were activated either by exposure to a low dose of carbachol (0.1 μM) in order to activate a store dependent contraction or by a high dose of carbachol (10 μM) where the contraction is more dependent on an influx of Ca²⁺. After treating the tissue with dilitiazem (10 μM), low and high doses of agonist were reapplied. (A single high dose of diltiazem was chosen since preliminary experiments showed that this dose produced a maximum effect on contraction). Contractions induced by low doses of carbachol (0.1 μM) were reduced to 29±11% (P<0.05) of controls while those activated by high concentrations (10 μM) were reduced to 86±6% (P=0.1) of controls (mean±s.e.mean; n=4). Thus, if an influx is essential to facilitate intracellular Ca²⁺ release at low doses of agonist it is likely to occur by diltiazem sensitive voltage operated Ca²⁺ channels. The absence of any significant effect of diltiazem at high agonist concentrations is consistent with the idea that in this dose range voltage operated channels do not contribute to the influx and that receptor operated channels or possibly other channels are primarily responsible. However at doses of 10 μM carbachol it has been suggested that carbachol may itself inhibit the inward Ca²⁺ current subsequent to depolarization and if so this could mask any effect of diltiazem (Yoshino & Yabu, 1995).

Figure 5.

The effect of the Ca²⁺ channel antagonist diltiazem on agonist induced contractions of human detrusor human smooth muscle. (A) Shows sections of original records from a single experiment. Where indicated carbachol at a concentration of either 0.1 μM (1) or 10 μM (2) was added to the superfusion solution. Also, where shown, diltiazem (10 μM) was added. Ordinate shows force produced by the muscle strip (mN) and the abscissa, time in s. All experiments were done at 35–37°C. (B) Illustrates data from four separate experiments presenting mean data of the amplitudes of contractions at two doses of carbachol in the absence (open bars) and presence (filled bars) of diltiazem (10 μM). Error bars show s.e. mean.

The results presented so far reflect the possibility that there is an absolute requirement for a rise in cytoplasmic Ca²⁺ in order to facilitate intracellular store release by IP₃ at low agonist concentrations. Such a co-operativity has been documented in many cell systems but it is not clear how it operates in human detrusor smooth muscle. For example, is Ca²⁺ required to facilitate IP₃ action or does IP₃ act to sensitize the release channel to Ca²⁺? In the latter mode the IP₃ receptor channel complex operates like an IP₃ modulated Ca²⁺ induced Ca²⁺ release mechanism. The data shown in Figure 6 support the idea that in the human detrusor the IP₃ receptor can operate as an IP₃ regulated CICR mechanism. Muscle strips were stimulated electrically to produce sub maximal contractions. Carbachol was then added to the superfusion solution at a concentration (0.03 μM) which did not by itself activate a contracture. In the presence of carbachol the amplitude of the electrically evoked contractions increased. This effect was seen in three out of four experiments. This augmentation of contraction was reversed on washing. These data are consistent with the idea that IP₃ is a co-factor in the process elevating Ca²⁺ and activating contraction.

Figure 6.

The effect of low dose agonist on the amplitude of electrically evoked contractions in human detrusor human smooth muscle. Ordinate shows force produced by the muscle strip (mN) and the abscissa, time in seconds. Where indicated, electrical stimuli (20 Hz; 20 V; 5 msec pulse width) was applied for periods of 2 s and carbachol (0.03 μM) was added to the superfusing solution. All experiments were done at 35–37°C.

Discussion

The major observation reported in this paper is that the intracellular Ca²⁺ stores contribute significantly to the rise in cytosolic Ca²⁺ needed to initiate contractions in human detrusor smooth muscle. This result on human detrusor smooth muscle is consistent with reports on a variety of smooth muscle types (Maggi et al., 1989; Neusser et al., 1993), and also on rabbit bladder smooth muscle which show that thapsigargin (Tg) treatment reduces the magnitude of agonist induced contraction (Damaser et al., 1997). An unexpected and new observation was that the effectiveness of Tg appeared to depend on the concentration of agonist used. One way to interpret these observations is to suggest that small contractions are activated using Ca²⁺ derived predominantly from the internal stores while large contractions rely almost entirely on an influx of Ca²⁺ from the external media. Therefore, if care is not taken to explore the effects of Tg over a suitable range of stimuli then it is possible that a false conclusion may be reached regarding the role and importance of intracellular store release and influx mechanisms. This also adds a further complication in the analysis of EC coupling in vivo where the precise stimulus and concentration of neurotransmitter may not be known.

At low concentrations of agonist the Tg data indicate that the internal stores are the predominant source of Ca²⁺ for contraction. However, the data presented in Figures 4 and 5 strongly suggest that this Tg sensitive intracellular release is highly dependent on an influx of external Ca²⁺. There are several possibilities which might account for this observation. It has been suggested that in some cell types the internal stores are depleted of Ca²⁺ at rest and it is only when the cells are stimulated resulting in an influx of Ca²⁺ that the stores fill and are subsequently able to release Ca²⁺ (Berridge, 1997). This may be the case here but the observation that high doses of agonist can release Ca²⁺ would argue against this. Alternatively, it may be that an influx of Ca²⁺ and a rise in cytoplasmic Ca²⁺ concentration is essential in order to facilitate IP₃ dependent release. This is in keeping with the positive feedback of Ca²⁺ on the IP₃ receptor channel complex. In the concentration range 100–300 nM, Ca²⁺ exerts a positive feed back on the release channels such that Ca²⁺ release is promoted. At cytoplasmic Ca²⁺ concentrations greater than 10 μM the IP₃ activated release channel is inhibited and Ca²⁺ cannot be released from the store (Bezprozvanny et al., 1991; Chambers et al., 1996; Iino & Endo 1992; Missiaen et al., 1994). Thus, the Ca²⁺ dependence of the IP₃ dependent Ca²⁺ release process can be described as ‘bell-shaped'. A rise in cytoplasmic Ca²⁺ is essential to sensitize the IP₃ receptor channel complex to low concentrations of IP₃ in order to initiate release. The data shown in Figure 6, demonstrating that low doses of agonist augment electrically induced contractions, are consistent with this mode of operation of the IP₃ Ca²⁺ release system. Electrical activity gives rise to a Ca²⁺ influx which can activate CICR and the combined changes in Ca²⁺ trigger contraction. In the presence of low levels of IP₃, the IP₃ receptor channel complex is primed but not yet activated. When the cytoplasmic Ca²⁺ rises this allows the IP₃ channel to open and Ca²⁺ to be released. The additional increase in Ca²⁺ from the stores augments the rise in cytoplasmic Ca²⁺ and increases the force of contraction.

The observations that, with Tg at high agonist concentrations, 8% of the Ca²⁺ comes from the stores while the experiments with nominally Ca²⁺ free solutions indicates that the stores can contribute 36% deserve comment. It would appear that in the presence of external Ca²⁺ the influx reduces the effective release of stored Ca²⁺. This is in keeping with the published data on the Ca²⁺ dependent inhibition of IP₃ dependent Ca²⁺release (Bezprozvanny et al., 1991; Chambers et al., 1996; Iino & Endo 1992; Missiaen et al., 1994).

It is clear from this analysis that an influx of Ca²⁺ is essential in order to activate the human detrusor smooth muscle. There are reports using L-type Ca²⁺ channel blockers, that cholinergic stimulation does not involve activation of voltage dependent Ca²⁺ channels and an influx of Ca²⁺ via this route (Fry & Wu, 1997). However, there are contrary reports suggesting that Ca²⁺ channel blockade does reduce contractions activated by agonists (Damaser et al., 1997; Fovaeus et al., 1987; Maggi et al., 1989). In the present experiments a distinct difference was noted in the action of Ca²⁺ channel blockers depending on the agonist concentration and the magnitude of the induced contraction. Small contractions are blocked by diltiazem but not large contractions. This implies that L-type Ca²⁺ channels are activated at low agonist concentrations. Additional Ca²⁺ influx pathways, possibly receptor operated Ca²⁺ channels or store regulated Ca²⁺ entry mechanisms, are activated at higher agonist concentrations. It is not possible, at this stage, to identify which might be responsible for this additional Ca²⁺ influx. At present, there are no estimates of the in vivo concentrations of agonist. Therefore, it is not possible to determine the events contributing to EC coupling in vivo. The situation may be more complex if more than one transmitter substance is co-released from nerve terminals in the bladder (Burnstock, 1985).

The interpretation of these data presented here are based upon the assumption that the magnitude of contraction is only related to the intracellular Ca²⁺ concentration. In rabbit detrusor when the contractile force and the intracellular Ca²⁺ have been measured simultaneously, then the increasing contractile response correlated with a nearly proportional increase in the magnitude of the free intracellular Ca²⁺ (Levin et al., 1991). However, in this study bethanechol stimulated contractions were completely blocked, a result which differs from that reported here. The reasons for these experimental differences are not clear but may be related to species differences. Other results have been described in rat detrusor where contractile responses to bethancol in various degrees of obstruction correlated with a nearly proportional increase in the magnitude of the free intracellular Ca²⁺ (Saito et al., 1994). No data are published for human detrusor and therefore we have presumed the results from these animal studies are applicable and that contraction in response to application of muscarininc agonists is dependent upon a proportional rise in the intracellular Ca²⁺ and proportional production in force. This is clearly an over simplification since it is now well recognized that there are a large number of complex intracellular signalling events between agonist binding and force development. However, the data presented in this paper do not totally rely on a detailed appreciation of these complexities. The major point to be made from this work is that the apparent effectiveness of Tg and hence the apparent contribution of intracellular Ca²⁺ release to contraction is related to the magnitude of the stimulus and correlates with the magnitude of contraction.

In addition, in smooth muscle, agonist stimulation can give rise to phenomenon of ‘Ca²⁺ sensitization' of the contractile apparatus (Somlyo & Somlyo, 1994). In relation to the data described in this paper of the effects of Tg the results cannot be explained in relation to Ca²⁺ sensitization. This is because, the carbachol doses are the same in both the absence and presence of Tg. However, the increase in contraction seen on the application of low dose agonist may also occur if the agonist were to sensitize the contractile apparatus to Ca²⁺ being elevated by the electrical activity. Subsequent to these interesting preliminary experiments, there is clearly a need to measure intracellular Ca²⁺ and tension simultaneously in human detrusor whilst repeating the experiments and this will be the focus of a future study.

Abbreviations

ACh

acetylcholine

EC

excitation contraction

IP₃

inositol trisphophate

RyR-CICR

ryanodine sensitive, Ca²⁺ induced Ca²⁺ release

SR

sarcoplasmic reticulum

Tg

thapsigargin