The effect of cyclopiazonic acid on excitation-contraction coupling in guinea-pig ureteric smooth muscle: role of the sarcoplasmic reticulum

Abstract

1. We have investigated the effect of cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase on excitation-contraction (EC) coupling in guinea-pig ureter, by measuring membrane currents, action potentials, intracellular [Ca²⁺] and force.

2. CPA (20 μm) significantly enhanced the amplitude and duration of phasic contractions of ureteric smooth muscle associated with action potentials. This was accompanied by an increase in the duration of the intracellular Ca²⁺ transient in intact tissue and single cells but not their amplitude. However, CPA also slowed the rate of rise, and fall, of the force and Ca²⁺ transients.

3. Membrane potential recordings showed that CPA produced a small depolarization and a large increase in the duration of the plateau phase of the action potential.

4. Patch-clamp studies showed marked inhibition of outward potassium current in the presence of CPA and an inhibition of spontaneous transient outward currents (STOCs). CPA had no effect on inward Ca²⁺ current.

5. These data suggest that the SR plays a major role in modulating the excitability of the ureter, particularly via curtailing the action potential duration. This in turn will shorten the Ca²⁺ transient and decrease force. This negative action on developed force predominates over any small role it may play in initiating force in the guinea-pig ureter.

The major mechanism responsible for an increase in force in smooth muscles is a rise in intracellular [Ca²⁺] ([Ca²⁺]_i). This rise occurs as Ca²⁺ enters the cell across the surface membrane and as Ca²⁺ is released from the internal store, the sarcoplasmic reticulum (SR). Ca²⁺ may be released from the SR by an IP₃-induced release mechanism or by Ca²⁺ itself, i.e. Ca²⁺-induced Ca²⁺ release (CICR) (Somlyo & Somlyo, 1994). Many smooth muscles use both mechanisms but some rely predominantly on either IP₃-induced release or CICR. The guinea-pig ureter has a purely CICR-type store (Burdyga et al. 1995, 1997). However, the importance of SR Ca²⁺ release for smooth muscle contraction, compared with surface membrane Ca²⁺ entry, has been questioned. For many smooth muscles it is reasonable to propose that Ca²⁺ entry is the major source of activating Ca²⁺ for contraction (Ganitkevich & Isenberg, 1995; Kamishima & McCarron, 1996; Taggart & Wray, 1998b).

Apart from supplying Ca²⁺ for contraction, the SR has been proposed to be an important mediator of electrical activity in smooth muscle and other tissues (Imaizumi et al. 1989, 1998; Nelson & Quayle, 1995). Spontaneous Ca²⁺ release from the SR activates Ca²⁺-sensitive surface membrane channels giving rise to currents (Carl et al. 1996). In some smooth muscles, both K⁺ and Cl⁻ channels activated by SR Ca²⁺ (K_Ca and Cl_Ca, respectively) have been identified and result in spontaneous transient outward and inward currents, STOCs and STICs, respectively (Carl et al. 1996). These currents will affect the membrane potential and hence affect Ca²⁺ entry through the voltage-gated L-type Ca²⁺ channels in the membrane (Large & Wang, 1996; Mironneau et al. 1996). In addition, emptying of the SR can activate capacitative Ca²⁺ entry via currents through calcium release activated channels (I_crac) (Missiaen et al. 1990; Byron & Taylor, 1995).

Thus the SR may contribute to excitation-contraction (EC) coupling in two ways, (i) release of Ca²⁺ for the myofilaments to initiate or augment contraction and (ii) modulation of membrane excitability. It is not therefore easy to predict the role and importance of the SR to EC coupling. One approach is to inhibit SR function using cyclopiazonic acid (CPA), a selective blocker of the SR Ca²⁺-ATPase in a number of cells (Goeger & Riley, 1989; Deng & Kwan, 1991) including ureter (Prischepa et al. 1996; Deng & Kwan, 1991). Maggi et al. (1995) had described a stimulatory action of CPA on guinea-pig ureter, but [Ca²⁺]_i was not measured and therefore the mechanism of the effect of CPA was unclear. The purpose of our study was therefore to systematically investigate the effect of inhibiting the SR using CPA on ureteric smooth muscle, and assess its role in EC coupling, by measuring electrical activity, [Ca²⁺]_i and force in the presence and absence of CPA.

METHODS

Tissue strips

Guinea-pigs (∼300 g) were anaesthetized with CO₂ and then killed by cervical dislocation. The ureter was dissected, cleared of any fat and cut into strips around 3–4 mm in length. For measurement of Ca²⁺, the ureter was incubated in the membrane-permeant form of indo-1 (15 μm; Molecular Probes) for 2–3 h at room temperature. Tissues were rinsed and then placed in a 200 μl bath on the stage of an inverted Nikon microscope. One end of the tissue was fixed and the other attached to a force transducer. The tissue was stimulated via silver electrodes, with 3–5 V, every 20–40 s (duration 50–100 ms). Action potentials were evoked by just-suprathreshold depolarizing current pulses of short duration (20–50 ms). For simultaneous force, Ca²⁺ and electrical measurements, a modified tissue bath was used, as detailed elsewhere (Burdyga & Wray, 1997). The sucrose-gap method was used to measure relative changes in membrane potential. Briefly the bath became a sucrose-gap chamber with a coverslip at its base, to enable the optical measurements to be made. For Ca²⁺ measurement the tissues were excited at 340 nm and the indo-1 fluorescence emitted at 400 and 500 nm recorded. The ratio of these signals (F_400/500) provides a measure of [Ca²⁺]_i (Grynkiewicz et al. 1985; Burdyga et al. 1995).

Single cells

Small pieces of ureter were incubated in an enzymatic dissociation solution as described previously (Sui & Kao, 1997; Smith et al. 1998). Briefly ureters were removed, cleaned of connective tissue under a dissecting microscope, placed in Ca²⁺-free Krebs solution and bubbled with 100 % O₂ for 30 min at 35°C. After that the lumen of both ureters was cut open and the epithelial cells were gently removed. Then the ribbons of the ureter were cut into small pieces 3–4 mm square and transferred into a digestion solution and incubated for 30–40 min at 35°C. The digestion solution contained collagenase (0.2 % w/v, 230 U min⁻¹; Worthington), soybean trypsin inhibitor (0.2 w/v, type II-S; Sigma) and fatty acid-free bovine serum albumin (0.6 %; Sigma). When the tissues appeared fluffy, they were removed from the digestion solution and washed through four to five transfers into Ca²⁺-free Krebs solution. After the last wash, the tissue was transferred to modified high-K⁺, low-Ca²⁺‘KB’ media (Sui & Kao, 1997), which consisted of (mM): 85 KCl, 30 K₂HPO₄, 5 MgSO₄, 5 Na₂ATP, 5 potassium pyruvate, 5 creatine, 20 taurine. The tissue was then gently triturated with several glass pipettes that had three heat-polished tip openings of 2–3, 1–2 and 0.5–1 mm. Suspensions of such dissociated cells were stored in the fridge before use when necessary. For recording total membrane currents, the pipette solution contained (mM): 140 KCl, 1 MgCl₂, 10 Hepes, 3 Na₂ATP, 1 (or 0.1) EGTA, 5 glucose (pH 7.3). For studying inward current, 140 mM Cs⁺ replaced K⁺ but all other constituents remained the same.

The whole-cell patch-clamp recording methods were applied to the isolated ureteric smooth muscle cells following previously described methods (Imiazumi et al. 1989; Lang, 1989; Sui & Kao, 1997). The recording system consisted of an Axopatch-1B CV4-1/100 patch-clamp amplifier, a dual-trace osciloscope, Digidata (analog-to-digital/digital-to-analog convertor) 1200 board. Most data acquisition was performed using pCLAMP software (Axon Instruments). The leakage currents at potentials positive to −60 mV were subtracted. Further data analysis and graphic plotting were done with a commercial pCLAMP module spreadsheet program (Origin 4.1). Whole-cell currents were recorded on-line at a sampling rate of 1 kHz and filtered through low-pass filters of 1–2 kHz.

Solutions

Tissues and cells were superfused with oxygenated buffered Krebs solution (pH 7.4) of the following composition (mM): 154 NaCl, 5.9 KCl, 1.2 MgSO₄, 2 CaCl₂, 11.5 glucose, 11 Hepes. Cyclopiazonic acid (20 μm) was obtained from Sigma. The experiments were performed at 35°C.

Statistics

Values are given as means ±s.e.m., and n is the number of animals or cells, as appropriate. Differences were taken as significant if P < 0.05 in the appropriate Student's t test.

RESULTS

The effect of CPA on ureteric Ca²⁺ and force

In these studies CPA was used as a tool to block SR function, by inhibiting the Ca²⁺-ATPase and depleting the SR of calcium. We have shown in earlier studies that CPA acts in this way: firstly, within 5 min CPA at 20 μm completely blocked ATP-dependent calcium accumulation into ureteric SR microsomes (Prischepa et al. 1996); secondly, our previous work on intact and permeabilized multicellular preparations of guinea-pig and rat ureter showed that, within 5 min, CPA emptied the SR, since caffeine or carbachol were no longer able to elicit a rise in Ca²⁺ or force (Burdyga et al. 1995, 1998). Hence CPA is assumed to act by depleting the SR of Ca²⁺ and thus functionally disabling it.

Figure 1 illustrates the typical changes (in 15 preparations) in Ca²⁺ and force produced by CPA in the guinea-pig ureter. CPA was normally applied for 10–20 min. As seen in Fig. 1A, CPA greatly potentiated the amplitude of the phasic contractions evoked by electrical field stimulation. The effect of CPA was fast and reached steady state within 4–5 min of administration. The amplitude of the phasic contraction was increased 1.86–3.20 times (n = 15). As also seen in Fig. 1A, the increase in the amplitude of force was not accompanied by an increase in the amplitude of the Ca²⁺ transient. However, CPA had a very strong influence on the duration of the Ca²⁺ transient. The duration of the Ca²⁺ transient, measured at 50 % amplitude after 4–5 min in CPA, increased from 730 ± 100 ms in control preparations to 2480 ± 80 ms in the presence of CPA (3.4 times; n = 15). There was an initial increase in the amplitude of the Ca²⁺ transient, although, unlike the force transient amplitude, this effect was not maintained. CPA also elevated basal Ca²⁺ but not basal force. Maximal changes in the basal level of [Ca²⁺], the amplitude and duration of Ca²⁺ transient and the amplitude and duration of phasic contractions took place within the first 5 min. After this there was a gradual recovery of basal level [Ca²⁺] (30–50 % after 20 min in CPA), and a decrease in the amplitude of the Ca²⁺ transient (20–30 %), in relation to control. Thus the increase in the force amplitude was associated with an increase in the duration of the Ca²⁺ transient but not its amplitude. These effects on Ca²⁺ and force transients can be seen more clearly in Fig. 1B, where specimen transients (i, ii and iii in Fig. 1A) are shown on an expanded time scale.

Figure 1. The effects of cyclopiazonic acid (CPA) on force and calcium.

A, the effect of CPA on the evoked phasic contractions and Ca²⁺ transients of the guinea pig ureter. B, phasic contractions and Ca²⁺ transients recorded in control conditions (i) and after 1.5 min (ii) and 5 min (iii) in CPA. C, the relationship between the amplitude of force and the duration of the Ca²⁺ transient. D, the phase plane diagram showing the force-Ca²⁺ relationship recorded during the development of the individual phasic contractions (see text).

The relationship between [Ca²⁺] and force is shown in Fig. 1D, where their amplitudes during the contraction are plotted. It can be seen that initially Ca²⁺ rises without an increase in force (A) and then there is a period when both force and Ca²⁺ increase (B). In control conditions this is followed by the decline of Ca²⁺. Force is, however, still increasing during a large portion of the time over which Ca²⁺ is falling (C) before eventually falling (D). Thus there is a hysteresis in the Ca²⁺-force relationship. When CPA is present this hysteresis is still seen but the ascending and descending limbs are closer together, as shown for traces ii and iii, in Fig. 1D. Thus the initial stages of the relationship are the same as under control conditions (Fig. 1D; Aand B), and then the Ca²⁺ is maintained with little or no further increase in the Ca²⁺ amplitude. During this period (E), force continues to rise and more force is produced than under control conditions (compare F and C). When [Ca²⁺] starts to decline in the presence of CPA (F) force follows earlier than under control conditions, hence G and D are shifted. This plot therefore suggests that force and Ca²⁺ are not in equilibrium during control conditions, but in the presence of CPA the prolongation of the Ca²⁺ transient extends the period over which force can develop, i.e. come into equilibrium with Ca²⁺ of its peak level (see Discussion).

From the above it would be expected that there should be a positive correlation between the duration of the Ca²⁺ transient around its peak amplitude, and the amplitude of the contraction. Figure 1C, which illustrates the relationship between the amplitude of force and the duration of the Ca²⁺ transient (measured at 50 % amplitude), shows that this was indeed the case. Within the range of 0.5–1 s this was almost linear.

Thus CPA significantly increases ureteric force predominantly by increasing the duration of the underlying Ca²⁺ transient.

Single cells and CPA

The above conclusions concerning force and Ca²⁺ were of necessity made on multicellular preparations, to permit the measurement of force. A concern with using such an approach might be that the Ca²⁺ changes measured may be unduly influenced by surface cells. In order to directly address this point, the effects of CPA on Ca²⁺ transients were examined in single isolated ureteric smooth muscle cells. The effects of CPA were very similar to those seen in multicellular preparations. As shown in Fig. 2 (typical of 5 other cells), CPA elevated basal [Ca²⁺], significantly increased the duration of the Ca²⁺ transients (from 400–700 ms in control to 1500–3100 ms in CPA) and produced only a small rise in Ca²⁺ transient amplitude (after subtraction of basal Ca²⁺). Thus the signals recorded in the multicellular preparations are correctly reporting the Ca²⁺ changes seen in individual cells. CPA also slowed the relaxation of the Ca²⁺ transient, as can be seen from the time constants (τ) of the monoexponential fitted to a control and CPA-affected Ca²⁺ transient. The quantitative data on this effect and that of CPA on Ca²⁺ transients and force are discussed in the next section, together with the data from intact tissues.

Figure 2. The effect of CPA on Ca²⁺ in single cells.

A, changes in intracellular [Ca²⁺] in a single ureteric cell, evoked by brief exposure (1 s) to high-K⁺ solution before and after addition of CPA. B, normalized Ca²⁺ transients in the absence and the presence of CPA, showing the monoexponential fittings to the decaying phase of the Ca²⁺ transients.

Effect of CPA on the kinetics of the Ca²⁺ and force transients

To examine whether the SR plays a role in the rate of rise or removal of [Ca²⁺]_i, which in turn may influence the temporal characteristics of the phasic contraction, the effect of CPA on force and Ca²⁺ transient kinetics was examined. Figure 3 shows normalized, superimposed Ca²⁺ and force transients in the presence and absence of CPA. On this expanded time scale, the slower rise of both the Ca²⁺ and force transients in CPA is apparent. The rising phase of the Ca²⁺ transients consisted of two parts; a fast initial one lasting 40–60 ms and a slower phase lasting 200–300 ms. The amplitude of these two components of Ca²⁺ rise were decreased in CPA (Fig. 3C). As a result the rate of rise of the overall Ca²⁺ signal during this period, measured as an integral, was 77 ± 11 % of control (n = 12). The mean rate of rise time was also substantially slower for force, being 2.08 ± 0.20 ms in CPA and 2.87 ± 0.15 ms in control (n = 12). These differences can be clearly seen in Fig. 3B, where the rate of change of force is shown for the data in Fig. 3A. Figure 3B also shows that CPA significantly slowed the rate of relaxation (1.53-fold slower). The time constant (τ) of the Ca²⁺ transient decay (fitted using a monoexponential) was increased significantly by CPA, from 0.26 ± 0.01 ms under control conditions to 0.73 ± 0.05 ms in cells (n = 6), and from 0.31 ± 0.06 to 0.71 ± 0.11 ms in tissues (n = 12). These data suggest that (i) the SR plays a role in the initial rise of Ca²⁺ which in turn, influences the rate of force production and (ii) the SR contributes to the restoration of the Ca²⁺ transient and relaxation of force.

Figure 3. The effect of CPA on kinetics of the Ca²⁺ transients and force.

A, superimposed records of the normalized Ca²⁺ transients and phasic contractions evoked by electrical field stimulation. B, rates of contraction and relaxation of the normalized phasic contraction recorded in the absence and the presence of CPA, showing the slower rates of both rise and fall produced by CPA. C, superimposed traces of the rising phase of the Ca²⁺ transients recorded in the presence and the absence of CPA.

The effect of CPA on the action potential

The above data show that both the kinetics and the duration of the Ca²⁺ transient are altered by CPA application to the guinea-pig ureter. It is known that Ca²⁺ entry is due to the action potential and that its modulation can alter [Ca²⁺]_i and force. In an earlier study, Maggi et al. (1995) had shown that CPA increased the duration of the plateau component of the ureteric action potential. In order to determine directly if there was a correlation between the changes produced by CPA on the action potential and the Ca²⁺ and force changes, we have made simultaneous measurements of all three parameters.

In Fig. 4A, one such (typical of 6) simultaneous record of force (top), [Ca²⁺] (middle) and membrane potential (E_m, bottom) is shown, in the absence and then presence of CPA (7 min application). It can be seen that CPA produced a prolongation of the action potential (from 400–600 ms in control to 1500–1800 ms in CPA) and that this was associated with a prolongation of the Ca²⁺ transient at 90 % peak level. This in turn was associated with the increase in the amplitude of the phasic contraction previously described. As can be seen in Fig. 4B, there was a good correlation between the duration of the action potential, measured at its 50 % level and the duration of the Ca²⁺ transient, measured at its 90 % peak value. In addition, CPA increased the amplitude of the spikes. In those preparations where spikes were poor under control conditions, CPA enhanced them. There was also a slight depolarization (3–5 mV) in the presence of CPA.

Figure 4. The effect of CPA on electrical activity.

A, simultaneous recording of the force (top trace), Ca²⁺ transients (middle trace) and action potentials (E_m; bottom trace) obtained in the absence and the presence of CPA. B, the relationship between the amplitude of force, the duration of the Ca²⁺ transient measured at its 90 % peak level (t_90%) and the duration of the action potential measured at its 50 % level (t_½)(n = 6).

The effect of CPA on ionic currents

Given that CPA significantly increases the plateau period of the guinea-pig action potential, as well as enhancing the spike component, and that these changes underlie those of Ca²⁺ and force, the question becomes, how is CPA exerting its effects on the action potential? There are two possibilities: firstly, that CPA is enhancing the inward Ca²⁺ current, through L-type Ca²⁺ channels, as these play a key role in the generation of the plateau component (Imiazumi et al. 1989; Lang, 1989; Sui & Kao, 1997); secondly, that CPA inhibits the calcium-activated K⁺ current (I_K,Ca), which is the major outward current in the guinea-pig ureter (Lang, 1989). Imiazumi et al. (1989) have shown that depletion of the SR Ca²⁺ store had a strong inhibitory effect on I_K,Ca current. However, the SR was depleted by caffeine, which, unlike CPA, shortened the duration of the action potential and decreased the amplitude of phasic contractions (Burdyga & Magura, 1986). Thus it is unclear what effect CPA would have on I_K,Ca in guinea-pig ureter. We have therefore used voltage-clamped single ureteric cells to examine the effect of CPA on membrane currents.

Figure 5 shows the effect of 20 μm CPA on the K⁺ outward current recorded in the presence of 1 mM EGTA in the pipette solution (typical of 12 cells). Normally, depolarizing steps elicited an initial inward current immediately followed by a large transient outward current (Fig. 6A). Application of 20 μm CPA markedly reduced this transient outward current and a small inward current could be unmasked (Fig. 6B). The effect of CPA was fully reversible (Fig. 5) and could be repeated several times in one cell (not shown). When long depolarizing steps were applied and the concentration of EGTA in the pipette solution was lowered to 0.1 mM, large STOCs were recorded in the guinea-pig ureteric cells (Fig. 6C). CPA (Fig. 6D) reversibly blocked these STOCs.

Figure 5. The effect of CPA outward currents of uretereal myocytes.

Clamping protocol: from a holding potential of −80 mV, membrane potential was clamped from −60 to +60 mV. A, control record; B, 5 min in CPA; C, 10 min after removal of CPA from the bath. The pipette solution had normal K⁺ concentration.

Figure 6. The effect of CPA on the initial transient outward current and total membrane currents.

The effect of CPA on the K⁺ outward current (A) and spontaneous transient outward currents (STOCs; C and D), recorded from single ureteric cells under voltage-clamp conditions. In A, the pipette solution contained 1 mM EGTA and the cell was depolarized from a holding potential of −80 mV to 0 mV. In C and D the pipette solution contained 0.1 mM EGTA, and the cell was depolarized from −80 mV to −20 mV. B, the current-voltage relationship recorded in the absence and the presence of CPA.

Addition of TEA (5 mM) to CPA-containing solution practically abolished the outward current and a large inward current was normally seen (Fig. 7; 7 cells). CPA did not affect the Ca²⁺ or Ba²⁺ inward current in the absence and the presence of Bay K, a Ca²⁺ channel agonist (Fig. 8). Thus CPA produced a selective inhibition of the STOCs, which reflects the spontaneous release of Ca²⁺ from the SR. Thus these data indicate the Ca²⁺ released from the SR is the main cause of the initial large outward current and that CPA selectively inhibits this current.

Figure 7. The effect of CPA and CPA plus TEA on the outward K⁺ currents.

From a holding potential of −80 mV, the membrane potential was clamped from −60 to +10 mV. A, control record; B, 5 min in CPA; C, 5 min after addition of TEA (5 mM) to the bath; D shows superimposed specimen records from A, B and C recorded during the depolarization to 0 mV. The pipette solution had normal K⁺ concentration.

Figure 8. The effect of CPA on the inward Ca²⁺ currents in the absence (A) and presence (B) of BayK (1 μm) and on the Ba²⁺ current (C).

From holding potential of −80 mV, the membrane potential was clamped from −60 to +60 mV. Pipette solution contained 140 mM Cs⁺. In A and B the bath media contained 2 mM Ca²⁺ and in C it contained 2 mM Ba²⁺. D-F, current voltage plots of inward current peaks vs. membrane potential recorded in 2 mM Ca²⁺ with Bay K and 2 mM Ba²⁺, respectively (in all plots □ are from control currents and • are current values recorded 5 min after addition of 20 μm CPA).

Effect of CPA on Ca²⁺ and force in the presence of TEA

The data presented suggest that CPA may inhibit only part of the Ca²⁺-activated K⁺ currents; that part activated by Ca²⁺ released from the SR. Since TEA blocks all I_K,Ca (Imiazumi et al. 1989; Lang, 1989; see also Fig. 8) it was of interest to determine the effect of TEA on the electrical activity, [Ca²⁺]_i and force recorded in the presence of CPA. Typical changes in these parameters recorded in the presence of CPA and CPA plus TEA are shown in Fig. 9. It can be seen that when CPA had produced its maximal effect, addition of TEA (5 mM) produced further increases in the amplitude and duration of the action potential. These were associated with a further increase in the duration of the Ca²⁺ transient accompanied by an additional increase in the amplitude and duration of the phasic contraction (Fig. 9). The action of TEA in the presence of CPA was to further increase the action potential and duration of the Ca²⁺ transient 1.4- to 2.0-fold and the amplitude of the contraction 1.15- to 1.30-fold, depending upon the effect of CPA (n = 9). In those preparations which showed moderate changes in the parameters of the action potential, calcium and force, in response to CPA, the average increase in the force amplitude was 1.56 ± 0.43 times. In the presence of both CPA and TEA the amplitude of force was increased 1.92 ± 0.44 times (n = 6). In the preparations where CPA produced a very marked stimulation, addition of TEA did not significantly alter the amplitude of force, although the duration of the Ca²⁺ transient and action potential were still significantly increased. Conversely, the stimulatory action of CPA was significantly attenuated when the preparations were pretreated with TEA (not shown).

Figure 9. The effects of CPA and CPA plus TEA on the parameters of the action potential.

The bottom record shows action potentials (E_m). Simultaneous recordings of Ca²⁺ transient (middle traces) and force (top traces) are also shown.

DISCUSSION

The data in this paper have helped elucidate the role of the SR in excitation-contraction coupling in ureteric smooth muscle. When the store is unable to take up Ca²⁺, due to inhibition of the SR Ca²⁺-ATPase by CPA, force is increased. The increase in the amplitude and duration of the phasic contractions in response to electrical stimulation was accompanied by a significant prolongation of the Ca²⁺ transient at maximal amplitude. The changes in Ca²⁺ transient could be attributed to the underlying action potential, which was also altered by CPA; the spike component increased and the plateau phase was prolonged. CPA produced no alteration of inward Ca²⁺ current but did inhibit K⁺ current and STOCs. Inhibition of Ca²⁺ uptake into the SR also led to an elevation of basal Ca²⁺, and a slowing of the relaxation of force and Ca²⁺. Finally CPA significantly slowed the rate of rise of Ca²⁺ and force. This suggests that SR Ca²⁺ plays only a small role in initiating contraction but that it can influence the membrane potential and excitability of ureteric smooth muscle to inhibit maintained contraction, as well as playing a role in Ca²⁺ sequestration and relaxation.

The functional role of the guinea-pig ureteric Ca²⁺ store was investigated by inhibiting the SR Ca²⁺-ATPase with CPA. At the concentration used (20 μm) CPA is a good pharmacological tool for selectively studying the SR (Uyama et al. 1993; Bourreau et al. 1993). The efficacy of CPA was seen in its elevation of basal Ca²⁺ and slowing of relaxation. Similar effects were seen in both intact tissues and single cells, demonstrating that effects in multicellular preparations are representative of those occurring in single cells. The effect on contraction - a marked potentiation - is perhaps puzzling since one might predict that removal of the SR and its contribution to Ca²⁺ release would reduce force, especially as previous work has demonstrated that this tissue contains a CICR mechanism (Burdyga et al. 1995, 1997). As stated earlier, the role of the SR in smooth muscle is 2-fold: to provide Ca²⁺ for the myofilaments and to affect electrical activity. Therefore in order to understand the contribution of the SR to force production it is necessary to examine its role and importance in both these processes in the ureter.

A role for the SR Ca²⁺ store in force production in the guinea-pig ureter?

The application of CPA to the guinea-pig ureter was previously found to deplete releasable Ca²⁺ from the SR (Burdyga et al. 1995). Despite the emptying of the Ca²⁺ store, there was no decrease in the Ca²⁺ transient amplitude or duration or force with CPA. This is in agreement with a previous study in the ureter (Maggi et al. 1995) where force (but not [Ca²⁺]) was measured. CPA has also been reported to enhance contraction in uterus (Taggart & Wray, 1998b) and vascular (Sekiguchi et al. 1996) and ileal (Uyama et al. 1993) smooth muscles. These results indicate that there can be little role for CICR in affecting either the magnitude of the transient Ca²⁺ or activating Ca²⁺ for the myofilaments, in these tissues. Similar conclusions were also drawn by Maggi et al. (1994), after using ryanodine, which prevents SR Ca²⁺ release by CICR, and finding no decrease in ureteric force. Evidence in favour of CICR playing a role in initiating contraction was presented in a recent confocal study of bladder and vas deferens, by Imaizumi et al. (1998). They concluded that Ca²⁺ entry in the early stages of an action potential evokes CICR from discrete sites, which generate hot spots and spread to initiate contraction. While we see no such role for the store, we did, however, see a small, but significant slowing of the rate of rise of [Ca²⁺] when CPA was present, suggesting that the SR may contribute to this rate of rise, and thus play a minor role in initiating contraction. Thus we conclude that, under physiological conditions, Ca²⁺ entry via L-type Ca²⁺ channels is the most important route for increasing [Ca²⁺] during the normal Ca²⁺ transient and phasic contraction of guinea-pig ureter.

CPA caused an elevation of basal Ca²⁺ and a slowing of recovery of the Ca²⁺ transient, consistent with a role for the SR in normally sequestering and maintaining low Ca²⁺ levels within the cell. A rise in basal Ca²⁺ upon inhibition of the SR Ca²⁺-ATPase with CPA has also been reported to occur in other smooth muscles, e.g. bladder (Munro & Wendt, 1994) and uterus (Taggart & Wray, 1998b), and also occurs during metabolic inhibition, when SR function may be impaired (Taggart & Wray, 1998a). Similarly the slowing of relaxation and Ca²⁺ restoration following cessation of stimulation is also consistent with a role for the SR Ca²⁺-ATPase taking up Ca²⁺. Clearly, however, this cannot be the only mechanism, as Ca²⁺ is still restored. Both Na⁺-Ca²⁺ exchange (Aickin et al. 1984; Aaronson & Benham, 1989), and the sarcolemmal Ca²⁺-ATPase (Prischepa et al. 1996) contribute to Ca²⁺ restoration after stimulation in the ureter, although the precise contribution of each under physiological conditions is unclear. It is also possible that the blockade of one mechanism, i.e. the SR re-uptake, leads to compensatory increases in other mechanisms (Becker et al. 1989).

The effects of CPA on electrical activity, [Ca²⁺]_i and force

The prolongation of the Ca²⁺ transient at near-maximal [Ca²⁺] (increased over 3-fold, compared with control conditions) was one of the most striking effects of CPA treatment. It is difficult to relate this, and the associated increase in force, to the SR operating just as a source and sink of Ca²⁺. Rather, as was shown by the electrophysiological data, the effects suggest an action of the SR Ca²⁺ on membrane currents, and, subsequently, Ca²⁺ entry, and that the SR acts to limit maintained contraction, once initiated. Effects on electrical activity following emptying of the SR have been suggested by other studies using CPA or thapsigargin, another inhibitor of the SR Ca²⁺-ATPase (Thastrup et al. 1990). For example, the increase in [Ca²⁺] and force following administration of these drugs has been shown to be sensitive to extracellular [Ca²⁺] in some (Xuan et al. 1992; Uyama et al. 1993), but not all (Baro & Eisner, 1992), cases, and effects on electrical activity have also been demonstrated (Suzuki et al. 1992; Maggi et al. 1995). Our voltage-clamp data in both multicellular and single cells, taken together with the force and Ca²⁺ measurements, suggest a large effect of CPA on outward current affecting the membrane potential, and hence Ca²⁺, and ultimately contraction, and therefore we will discuss this next.

The opening of voltage-gated L-type Ca²⁺ channels occurs during the action potential. The maintenance of this entry will depend, therefore, upon the membrane potential remaining depolarized and a slow rate of channel inactivation. The action potential of the guinea-pig ureter is very characteristic, as it consists of an initial fast depolarizing spike, followed by some repolarization and a further series of spikes and a long plateau phase, which may last for 300–1000 ms before final repolarization (Shuba, 1981). Single-cell studies showed that the inward current was due to Ca²⁺ entry and that the outward currents were largely Ca²⁺-activated K⁺ channels (Lang, 1989; Imaizumi et al. 1989; Sui & Kao, 1997). Furthermore, in the ureter, a large portion of the Ca²⁺ that activates the K⁺ channel arises from the SR (Imiazumi et al. 1989). Consistent with the long plateau phase of the action potential, the Ca²⁺ channel of the ureter has a very slow rate of inactivation, compared with uterine or intestinal Ca²⁺ currents, for example (Kao, 1997). Periods of slow Ca²⁺ channel inactivation followed by rapid reactivation are thought to contribute to the repetitive spikes on the action potential, along with repetitive activation of the K_Ca channels due to Ca²⁺ release from the store. These K⁺ channels, activated during the plateau phase of the action potential, lead eventually to hyperpolarization, and thus curtail the action potential and Ca²⁺ entry. Inhibition of K_Ca channels, e.g. by TEA, charybdotoxin or iberiotoxin, is associated with prolongation of the action potentials (Kao, 1997). Spontaneous Ca²⁺ release from the SR can also strongly affect K_ca channel activity, leading to STOCs.

From the above it seems likely that when the store is emptied, by CPA or similar drugs, this source of activating Ca²⁺ for the K_Ca channel is removed and hence action potential duration is increased. As shown here, this prolongation leads to the maintenance of the Ca²⁺ transient. In addition, the small depolarization seen in the presence of CPA, also reported by Maggi et al. (1995), suggests a contribution of K_Ca current to the membrane potential. We found a very obvious effect of CPA on STOCs and outward current. Stenho-Bittel & Sturek (1992) in vascular myocytes also reported that SR Ca²⁺ release contributes to both STOCs and steady-state K⁺ current. This stimulation of K⁺ channels may or may not be similar to the Ca²⁺ spark events which underlie STOCs (Nelson & Quayle, 1995). Upon reduction of the normal outward current a small inward current was unmasked, which may have been due to Na⁺ (Imaizumi et al. 1989). The sensitivity of these currents to TEA also supports the hypothesis that the K_Ca channels are the target of CPA action; prior treatment with TEA or CPA greatly reduced the subsequent action of the other, although further characterization of the channel involved is necessary to confirm this. CPA was, however, specific in reducing the outward current, since it was without effect on Ca²⁺ or Ba²⁺ inward current.

When the repolarizing effect of K⁺ current was reduced and the plateau phase of the action potential prolonged by CPA, the Ca²⁺ transient was also prolonged. We will now discuss the relationship between the Ca²⁺ transient and force in the ureter.

Ca²⁺-force relationship in guinea-pig ureter

CPA produced only a transient increase in the amplitude of the Ca²⁺ transient, but consistently produced a maintained increase in its duration. These changes were correlated, in simultaneous recordings, with a maintained increase in both the amplitude and duration of the phasic contractions. It might be expected that the increase in contraction amplitude should be mirrored by an increase in Ca²⁺ transient amplitude. The explanation for this discrepancy becomes clear when the kinetics of the relationship are examined, as shown in Fig. 1D. There is a hysteresis in the Ca²⁺-force relationship - Ca²⁺ and force are not in equilibrium during the normal phasic contraction. Thus the slower myofilaments are still producing force when the brief Ca²⁺ transient has been terminated and Ca²⁺ is declining. In CPA, because the Ca²⁺ transient has been prolonged, at near-maximal [Ca²⁺], the period over which force can develop and come closer to an equilibrium with Ca²⁺ is extended. Thus greater levels of force are obtained, without further increase in [Ca²⁺]. This lack of steady state during the phasic contraction of ureteric (and other smooth muscles) means that manipulation of the action potential, and hence Ca²⁺ transient characteristics, is an important physiological mechanism for altering force production (T. V. Burdyga & S. Wray, unpublished observations).

Since CPA enhanced force production in the ureter, this suggests that the normal activity of the SR is to depress force production. This it does by affecting membrane excitability, specifically by releasing Ca²⁺ which activates outward currents and repolarizes the tissue. This in turn stops Ca²⁺ entry via voltage-gated L-type Ca²⁺ channels and hence as Ca²⁺ is reduced so too is force. Thus in the ureter, control by the store of electrical activity and thereby an inhibition of contraction once initiated, appears to be its major function.