The role of the L-type Ca²⁺ channel in refilling functional intracellular Ca²⁺ stores in guinea-pig detrusor smooth muscle

Abstract

The transient rise of intracellular Ca²⁺ in detrusor smooth muscle cells is due to the release of Ca²⁺ from intracellular stores. However, it is not known how store refilling is maintained at a constant level to ensure constancy of the contractile response. The aim of these experiments was to characterise the role of L-type Ca²⁺ channels in refilling. Experiments used isolated guinea-pig detrusor myocytes and store Ca²⁺ content was estimated by measuring the magnitude of change to the intracellular [Ca²⁺] ([Ca²⁺]_i) after application of caffeine or carbachol using epifluorescence microscopy. Membrane potential was controlled when necessary by voltage clamp. After Ca²⁺ stores were emptied they refilled with an exponential time course, with a time constant of 88 s. The value of the time constant was similar to that of the undershoot of [Ca²⁺]_i following store Ca²⁺ release. The degree of store filling was enhanced by maintained depolarisation, or by transient depolarising pulses, and attenuated by L-type Ca²⁺ channel antagonists. Inhibition of the sarcoplasmic reticular Ca²⁺-ATPase prevented refilling. Reduction of the resting [Ca²⁺]_i was accompanied by membrane depolarisation; under voltage clamp reduction of [Ca²⁺]_i decreased the number and magnitude of spontaneous transient outward currents. Ca²⁺ release from intracellular stores, elicited by caffeine or carbachol, is independent of membrane potential under physiological conditions. However, store refilling occurs via Ca²⁺ influx through L-type Ca²⁺ channels. Ca²⁺ influx is regulated by a feedback mechanism whereby a fall of [Ca²⁺]_i reduces the activity of Ca²⁺-activated K⁺ channels, causing cell depolarisation and an enhancement of L-type Ca²⁺ channel conductance.

The motor drive to the urinary bladder is predominantly via postganglionic parasympathetic nerves (Brading, 1987). In normal human detrusor muscle contraction is mediated exclusively by acetylcholine binding to muscarinic M₃ receptors on the cell membrane. Their activation initiates the generation of inositol trisphosphate (Iacovou et al. 1990), a rise of the intracellular Ca²⁺ ([Ca²⁺]_i) (Fry et al. 1994) and muscle contraction (Sibley, 1984).

With detrusor from most animals, and also human tissue under certain pathological conditions such as detrusor instability (Bayliss et al. 1999), ATP acts as a secondary cotransmitter by depolarising the muscle cell. Detrusor smooth muscle is capable of generating action potentials (Creed et al. 1983; Brading & Mostwin, 1989) with the upstroke supported by Ca²⁺ influx through L-type Ca²⁺ channels (Klockner & Isenberg, 1985; Montgomery & Fry, 1992) and which is sufficient to cause a significant rise of [Ca²⁺]_i (Ganitkevich & Isenberg, 1991; Wu & Fry, 1998). However, the physiological role of the action potential and the L-type Ca²⁺ channel in detrusor contractile function is not clear, especially as normal human detrusor muscle can function purely by muscarinic receptor activation and independent of changes to the membrane potential. Ca²⁺ influx may play a role in generating spontaneous contractions observed in detrusor muscle from unstable bladders (Kinder & Mundy, 1987; Wu et al. 1997) and also mediate the contractile action of purinergic neurotransmitters (Hashitani & Suzuki, 1995; Wu et al. 1999a). However, the role of electrophysiological processes in the generation and maintenance of normal detrusor contractions initiated by cholinergic activation is not understood.

It has been suggested that cross-talk between electrophysiological systems and second messenger pathways may exist in some smooth muscles (Somlyo & Somlyo, 1994). However, little work has been carried out on the involvement of the Ca²⁺ channel in cholinergic pathways. Ca²⁺ channel antagonists or removal of extracellular Ca²⁺ attenuate carbachol-induced contractions, although to a lesser extent than their actions on high K⁺ or ATP-induced contractions (Mostwin, 1985; Maggi et al. 1988; Kishii et al. 1992). However, it does suggest an involvement of the Ca²⁺ channel in the contraction mediated by cholinergic activation, although these experiments gave no insight into the mode of action. In addition, Ca²⁺ channels may also be involved in replenishing intracellular Ca²⁺ stores, following Ca²⁺ release by neurotransmitters, to maintain the cycling of Ca²⁺ in response to repetitive stimuli. Experiments using contraction as an index of the size of such internal Ca²⁺ stores have shown that in tracheal smooth muscle, the L-type Ca²⁺ channel is partially involved in the refilling of acetylcholine-sensitive Ca²⁺ stores (Bourreau et al. 1991). Direct measurement of intracellular Ca²⁺ has also shown that the Ca²⁺ channel plays a role in caffeine-sensitive stores in coronary artery smooth muscle (Sturek et al. 1992). However, such a role of the Ca²⁺ channel in a smooth muscle such as detrusor that contracts phasically (Rivera et al. 1998) has not been examined.

The present study was undertaken firstly to clarify the role of the L-type Ca²⁺ channel in initiating the rise of intracellular Ca²⁺ associated with contraction, and secondly to investigate its role in refilling intracellular Ca²⁺ stores in cholinergic pathways. Our results show that the primary activation of cholinergic pathways is independent of membrane potential and the opening of the L-type Ca²⁺ channel, but can be modulated by secondary voltage-dependent membrane events. More importantly, the L-type Ca²⁺ channel may play a role in maintaining Ca²⁺ entry and refilling functional intracellular Ca²⁺ stores.

METHODS

Cell isolation

Single detrusor smooth muscle cells were obtained from guinea-pig urinary bladders. Guinea-pigs (400–900 g) were killed by cervical dislocation or concussion in accordance with current UK Home Office procedures. The bladder was quickly removed and placed in Ca²⁺-free solution containing (mm): NaCl, 105.4; NaHCO₃, 20.0; KCl, 3.6; MgCl₂, 0.9; NaH₂PO₄, 0.4; glucose, 5.5; sodium pyruvate, 4.5; Hepes, 4.9; pH 7.1. Small strips of detrusor muscle were cut from the dome of the bladder and cells were dissociated using a collagenase-based enzyme mixture dissolved in Ca²⁺-free solution as described previously (Montgomery & Fry, 1992).

Solutions

Myocytes were continuously superfused with Tyrode solution containing (mm): NaCl, 118; NaHCO₃, 24.0; KCl, 4.0; MgCl₂, 1.0; NaH₂PO₄, 0.4; CaCl₂, 1.8; glucose, 6.1; sodium pyruvate, 5.0; pH 7.4, gassed with 95 % O₂ and 5 % CO₂ at 37 °C. Zero-Ca²⁺ superfusate contained no added CaCl₂ and 0.1 mm EGTA, which buffered the ionised Ca²⁺ concentration to approximately pCa 8. The intracellular filling solution for patch pipettes contained (mm): KCl, 20; aspartic acid, 100; MgCl₂, 5.45; Na₂ATP, 5.0; Na₃GTP, 0.2; EGTA, 0.05; Hepes, 5.0; pH 7.1 adjusted with KOH.

Measurement of [Ca²⁺]_i

[Ca²⁺]_i was measured by epifluorescence microscopy using the fluorescent Ca²⁺ indicator fura-2. Cells were loaded with 5 μm fura-2 acetoxymethyl (AM) ester at 25 °C for 30–60 min and then stored at 4 °C for later use. An aliquot of cell suspension was placed in a chamber mounted on the stage of an inverted microscope and superfused at 37 °C with Tyrode solution. The cells were illuminated alternately at 340 and 380 nm. The emitted light was split by a dichroic mirror centred at 410 nm, and collected by a photomultiplier between 410 and 510 nm.

The fura-2 ratio signal was converted to [Ca²⁺]_i values using an in vitro calibration method (eqn (1)). The relationship between [Ca²⁺]_i and the ratio R (fluorescence ratio at 340 nm/380 nm excitation) is given by (Grynkiewicz et al. 1985):

(1)

where R is the fluorescence ratio under study, R_min and R_max refer to ratio values at zero [Ca²⁺] and saturating [Ca²⁺], respectively, β is the ratio at zero and saturating [Ca²⁺] at 380 nm alone, and K_d (224 nm) is the dissociation constant of fura-2 for Ca²⁺.

Electrophysiological recordings

Membrane potential and ionic currents were recorded with patch-type electrodes in the whole-cell configuration. An Axopatch-1D amplifier system (Axon Instruments) was used to perform current or voltage clamp. An IBM-compatible computer was used to generate voltage-clamp protocols and record membrane currents via an A/D converter (Digidata 1200; Axon Instruments) with a sampling frequency of 4 kHz and cut-off frequency of 2 kHz. Voltage-clamp protocols were supported by pCLAMP software (Axon Instruments). Patch pipettes were made from borosilicate glass (1.5 mm o.d./0.8 mm i.d.) and had a resistance of 3–5 MΩ when filled with the above intracellular solution. For simultaneous recording of [Ca²⁺]_i, cells were dialysed with fura-2 via a patch pipette filled with the intracellular solution containing 100 μm K₅-fura-2 (Calbiochem). This gave a sufficient signal-to-noise ratio without a significant effect on the Ca²⁺ transient (Wu & Fry, 1998).

In some experiments, high resistance (20 MΩ) microelectrodes were used to impale the cell and record the membrane potential without dialysing the intracellular space. Microelectrodes were filled with a potassium aspartate solution containing 1000 mm aspartic acid and 35 mm KCl; pH 7.1 with KOH. These allowed voltage following to be performed with a similar time resolution to that of conventional 3 m KCl-filled microelectrodes, but with a more stable cell penetration. The tip and junction potentials were nulled before cell impalement and remained stable throughout the experiment: the KCl was included to minimise disturbance of the intracellular [Cl⁻]. Membrane potential was recorded by a unit gain headstage (HS-2), connected to an Axoclamp-2A microelectrode clamp amplifier (Axon Instruments).

Muscle preparations and measurement of isometric tension

Mucosa and serosa were dissected from the bladder and a thin strip of muscle (< 0.4 mm in diameter, 5–6 mm in length) was carefully was cut using a binocular dissection microscope. The muscle strip was tied at one end to a fixed hook and at the other to an isometric tension transducer. The preparation was superfused with Tyrode solution in a horizontal trough (4 mm × 4 mm × 15 mm long) at a rate of 5–10 ml min⁻¹. Muscles were stimulated by addition of agonists to the superfusate. The tension transducer output was amplified and filtered at a cut-off frequency of 15 Hz. The signal was displayed on a storage oscilloscope and down-loaded to a pen recorder.

Statistics

Numerical data are presented as means ± s.e.m. Student's t test was used to test the significance of difference between two data sets of equal variance, and the modified t test used for two data sets of unequal variance. The null hypothesis was rejected when P < 0.05.

RESULTS

Characterisation of intracellular Ca²⁺ transients induced by cholinergic activation

Figure 1A shows that, in an isolated myocyte, the acetylcholine analogue carbachol (20 μm) transiently increased the [Ca²⁺]_i. The Ca²⁺ transient was preceded or coincided with a hyperpolarisation in 85 % (24 of 28) of cells, followed by a small depolarisation (8.8 ± 1.3 mV, 26 of 28 cells) that always occurred after the peak of the Ca²⁺ transient. Such a pattern is consistent with the induction of Ca-activated outward and inward currents due to a rise of [Ca²⁺]_i. A qualitatively similar pattern of changes to [Ca²⁺]_i and membrane potential occurred if caffeine was used to generate the Ca²⁺ transient. It has been demonstrated previously (Wu & Fry, 1998), and confirmed here, that a short (60 s) pre-exposure to an L-type Ca²⁺ channel antagonist (5–10 μm nifedipine or 20 μm verapamil) did not significantly affect the magnitude of this Ca²⁺ transient (98.3 ± 5.5 % of control, n = 8, P > 0.05), although this protocol reduced peak inward Ca²⁺ current by more than 80 % and abolished ATP-dependent Ca²⁺ transients that are elicited by membrane depolarisation.

Figure 1. Carbachol- and caffeine-induced intracellular Ca²⁺ transients.

A, intracellular [Ca²⁺] ([Ca²⁺]_i) and membrane potential (E_m) in an isolated detrusor myocyte. The horizontal dashed line indicates the time of peak hyperpolarisation; the vertical line indicates the E_m value prior to carbachol additon. B, carbachol (20 μm) induced Ca²⁺ transients in the absence and presence of 50 μm TMB-8 (the agents were added as indicated by the horizontal bars). C, caffeine (20 mm) induced Ca²⁺ transients in the absence and presence of 50 μm TMB-8.

Carbachol induces the Ca²⁺ transient by eliciting Ca²⁺ release from an intracellular Ca²⁺ store that is substantially overlapped by a caffeine-sensitive Ca²⁺ store (Wu & Fry, 1998). However, the mechanism for triggering Ca²⁺ release by carbachol is different from that by caffeine, as shown by the effect of 8-(N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl (TMB-8), which preferentially inhibits inositol trisphosphate (IP₃)-mediated release from intracellular Ca²⁺ stores (Anderson et al. 1992; Haughey et al. 1999). In the presence of 50 μm TMB-8 HCl, the carbachol-induced Ca²⁺ transient was effectively abolished (n = 4, see Fig. 1B), whilst caffeine Ca²⁺ transients persisted (87 ± 7 % of control, P > 0.05, n = 5, Fig. 1C). This suggests that carbachol generates Ca²⁺ transients via the IP₃ cascade, a mode of action not shared by caffeine.

It has been demonstrated in vascular smooth muscle that IP₃-induced Ca²⁺ release (IICR) itself may be modulated by membrane potential (Ganitketvich & Isenberg 1993). However, in the detrusor cells varying membrane potential (E_m) between −80 and +20 mV did not alter the magnitude of the Ca²⁺ transient. These data show that IICR in detrusor muscle is not facilitated by membrane depolarisation and that L-type Ca²⁺ channels play no role in generation of the Ca²⁺ transient. Further experiments were carried out to identify the nature of the Ca²⁺ store.

Characteristics of intracellular Ca²⁺ store refilling

The intracellular Ca²⁺ store can be depleted completely by a maximal effective concentration of either carbachol (20 μm) or caffeine (10–20 mm). Figure 2A shows that a rapid second exposure to either carbachol or caffeine after an initial carbachol intervention did not elicit a second Ca²⁺ transient. However, Fig. 2B shows that as the interval between paired exposures (in this case to caffeine) was increased, the Ca²⁺ transient gradually recovered. Attenuation of the second response was not due to receptor desensitisation, as caffeine and carbachol were equally ineffective in generating the second transient, regardless of the agonist used to generate the first response. Thus although carbachol and caffeine release intracellular Ca²⁺ via different mechanisms, both agonists seem to act on the same store with equal effectiveness. In the following experiments Ca²⁺ store properties are explored using both agonists.

Figure 2. Depletion and the time course of restoration of intracellular Ca²⁺ stores.

A, exposure to two applications of carbachol (20 μm) in rapid succession (left) or carbachol followed by caffeine (20 mm; right). Exposure periods are shown by the horizontal bars. B, paired exposures to caffeine (20 mm) at different intervals as indicated by the horiontal bars. C, plot of the recovery of the caffeine transient as a function of interval (t) between paired exposures. The data are plotted as a percentage of the second vs. first Ca²⁺ transient magnitude (% recovery) for each pair. Values are means ± s.e.m., n = 7. The line is a least-squares fit using the equation: %Recovery = 100(1 – exp(-(t–t₀)/τ)). τ is the time constant of the curve; the curve has been extrapolated to intersect the abscissa at t₀.

Figure 3C shows the mean data for the restoration of the maximal caffeine-evoked Ca²⁺ transient, fitted to an exponential function (eqn (2)):

(2)

where t₀ is a constant representing the intersection of the curve with the abscissa, corresponding to an absolute ‘refractory’ period when caffeine cannot trigger any Ca²⁺ release from the store (15.8 ± 4.1 s, n = 7). The restitution time constant (τ) for refilling the store was 88 ± 16 s (n = 7).

Figure 3. Carbachol-induced Ca²⁺ transients in zero-Ca²⁺ solution.

A, Ca²⁺ transients in 1.8 mm and zero-Ca²⁺ superfusates; there were three exposures to either carbachol or caffeine during the zero-Ca²⁺ superfusion. B, Ca²⁺ transients in 1.8 mm and zero-Ca²⁺ superfusates; a single carbachol exposure was introduced at the end of the zero-Ca²⁺ superfusion. C, Ca²⁺ transients in 1.8 mm and zero-Ca²⁺ superfusates and return to 1.8 mm Tyrode solution. In each trace carbachol exposure is indicated by the horizontal bars.

Depletion of the Ca²⁺ store was produced by removal of extracellular Ca²⁺ and the store could not be refilled in its absence. Figure 3A shows that during superfusion with a zero-Ca²⁺ solution, the releasable Ca²⁺ was gradually reduced, and second and third exposures to carbachol or caffeine elicited no further response: note also reduction of the resting [Ca²⁺]_i in this solution. Even with no intermediate agonist application (Fig. 3B), a prolonged exposure to zero-Ca²⁺ solution completely depleted the Ca²⁺ store. This suggests an exchange of Ca²⁺ between extracellular and intracellular spaces, as well as between the sarcoplasm and the intracellular Ca²⁺ store. Figure 3C shows that upon restoration of extracellular Ca²⁺, the resting [Ca²⁺]_i recovered and the response to agonists was completely restored (110 ± 6 % of pre-depletion level, n = 7) with a time constant (τ) of 112 ± 35 s. This τ value was not statistically different from the restitution value measured above with paired agonist exposures (Fig. 2C).

Membrane depolarisation enhances Ca²⁺ store refilling

Refilling of functional Ca²⁺ stores after zero-Ca²⁺ superfusion was facilitated by membrane depolarisation. Figure 4A shows the effect of raising the extracellular K⁺ concentration ([K⁺]_o). After a sufficient period to allow the Ca²⁺ transient to restore on return to a normal Ca²⁺-containing superfusate, a myocyte was exposed to carbachol. Figure 4A shows two superimposed carbachol-induced Ca²⁺ transients: one in a control [K⁺] (4 mm), and a second in a 24 mm K⁺ solution. Control carbachol-induced Ca²⁺ transients were similar to the pre-depletion transient (104 ± 9 %, P > 0.05, n = 9). In the 24 mm K⁺ solution the resting [Ca²⁺]_i was restored to a higher level compared with that in the 4 mm K⁺ solution, and moreover the carbachol-induced Ca²⁺ transient was significantly increased (135 ± 9 %; P < 0.05, n = 9). These data show that the quantity of Ca²⁺ sequestered in functional intracellular Ca²⁺ stores is related to the availability of sarcoplasmic Ca²⁺.

Figure 4. Membrane depolarisation and Ca²⁺ store refilling.

A, carbachol-induced Ca²⁺ transients before and after zero-Ca²⁺ solution. Two Ca²⁺ transients are superimposed after the zero-Ca²⁺ solution, in 1.8 mm Tyrode solution containing either 4 mm or 24 mm KCl. B, a similar format to A in which the cell membrane potential (E_m) was held under voltage clamp. Two carbachol-induced Ca²⁺ transients are superimposed after zero-Ca²⁺ solution: one after the cell was briefly depolarised to −25 mV, the other when E_m was held constant at −60 mV throughout. C, carbachol-induced Ca²⁺ transients in a detrusor cell under voltage clamp in Ca²⁺-containing Tyrode solution throughout. The cell was briefly depolarised from −60 to −25 mV between the second and third transients.

To control E_m more precisely in the refilling period after zero-Ca²⁺ superfusion, experiments were performed under voltage-clamp, when E_m was held at either −60 mV (near the normal resting potential) or at −25 mV (near the peak window current value for the L-type Ca²⁺ current as determined in control experiments). Figure 4B shows that a similar result was obtained to that in Fig. 4A: at −25 mV the carbachol-induced Ca²⁺ transient was enhanced to 146 ± 15 % (P < 0.05, n = 5) of that when the cell was held throughout at −60 mV. The value of E_m on replenishment of the carbachol-sensitive Ca²⁺ store was also important during continuous superfusion in Ca²⁺-containing Tyrode solution. Figure 4C shows that depolarisation from −60 to −25 mV enhanced the resting [Ca²⁺]_i as well as the carbachol-induced Ca²⁺ transient elicited immediately after repolarisation back to −60 mV (127 ± 14 % of control, P < 0.05, n = 15). Identical results were obtained if caffeine was used to generate Ca²⁺ transients; depolarisation from −60 to −25 mV enhanced refilling to 125 ± 7 % of control (P < 0.05, n = 8).

In normal extracellular [Ca²⁺], prolonged superfusion with 5 mm NiCl₂ reversibly abolished the caffeine-induced Ca²⁺ transient. As Ni²⁺ blocks membrane Ca²⁺ channels the role of such pathways in refilling was further investigated.

L-type Ca²⁺ channel manipulation and Ca²⁺ store refilling

Figure 5 shows an experiment when 10 μm nifedipine was added immediately after a carbachol-induced Ca²⁺ transient and throughout the interval before the next exposure to the agonist. The subsequent Ca²⁺ transient was significantly attenuated (47 ± 14 % of control, P < 0.05, n = 7). The interval between successive exposures allowed complete restitution in the absence of nifedipine as seen by the first two Ca²⁺ transients. Identical results were obtained if caffeine was used to elicit Ca²⁺ transients; they were reduced to 49 ± 5 % of control (P < 0.05, n = 9).

Figure 5. The L-type Ca²⁺ channel and Ca²⁺ store refilling.

A, carbachol-induced Ca²⁺ transients. Nifedipine (10 μm) was introduced between the second and third Ca²⁺ transients. B, carbachol-induced Ca²⁺ transients before and after zero-Ca²⁺ superfusate. Following the zero-Ca²⁺ solution verapamil (20 μm) was added to the Ca²⁺-containing superfusate for the first five carbachol exposures, thereafter the verapamil was removed. C, caffeine-induced Ca²⁺ transients in a detrusor cell under voltage-clamp at −60 mV. Between the first and second exposures to caffeine the cell was subjected to a train of five depolarising steps (-60 to +10 mV, 500 ms duration).

Figure 5B shows the effect of verapamil (20 μm) on the refilling of Ca²⁺ stores after depletion in a zero-Ca²⁺ solution. When the extracellular Ca²⁺ was restored in the presence of verapamil, the level of refilling did not return to the control value (65 ± 10 % of control, n = 7, P < 0.05). Complete recovery was only seen when verapamil was removed from the extracellular solution.

These data suggest involvement of L-type Ca²⁺ channels in refilling intracellular Ca²⁺ stores. However, T-type Ca²⁺ channels have also been described in detrusor smooth muscle (Sui et al. 2001) and it may be possible that the Ca²⁺ antagonists may also block these ion channels. However, the concentrations of antagonists used were chosen to be less than those reported to influence T-type channel conductance (Stengel et al. 1998). Moreover, their actions were similar in the continuous presence of 0.2 mm NiCl₂, which is sufficient to selectively block the T-type current in these cells. Nifedipine (10 μm) still reduced the carbachol and caffeine Ca²⁺ transients (52 ± 10 % of control, n = 3, P < 0.05) and by a magnitude was not significantly different from that in the absence of NiCl₂.

Conversely, opening L-type Ca²⁺ channels should enhance Ca²⁺ store refilling. Cells were voltage clamped at −60 mV and a train of five depolarising pulses from −60 to +10 mV for 500 ms was applied during the refilling interval. Figure 5C shows that a second application of caffeine after the stimulation procedure enhanced the Ca²⁺ transient (127 ± 8 % of control, n = 18, P < 0.01). A similar result was obtained if carbachol was used to evoke Ca²⁺ transients (119 ± 8 % of control, n = 7, P < 0.05).

Refilling via L-type Ca²⁺ entry is dependent on the sarcoplasmic reticulum Ca²⁺ pump

The exact route whereby Ca²⁺ enter intracellular Ca²⁺ stores is unclear. It is possible that the superficial sarcoplasmic reticulum (SR) is filled directly, by routes that may or may not involve the SR Ca²⁺-ATPase (SERCA), and that a rise of the bulk sarcoplasmic [Ca²⁺] is unnecessary (Casteels & Droogmans, 1981; Missiaen et al. 1990; Chen & van Breemen, 1993). However, the results of the present study are not compatible with this mechanism as the magnitude of Ca²⁺ store refilling is linked to similar changes of the bulk sarcoplasmic [Ca²⁺]. Lowering [Ca²⁺]_i diminished releasable Ca²⁺ from the stores, and restoration of the [Ca²⁺]_i after a zero-Ca²⁺ superfusate preceded the recovery of store releasable Ca²⁺ (Fig. 3). An alternative route therefore is that Ca²⁺ enters the sarcoplasm and is then pumped by the SERCA into the SR. To test this, depolarising pulses were applied in the presence of the SERCA inhibitor, cyclopiazonic acid (CPA). Figure 6 shows that 20 μm CPA attenuated the first caffeine Ca²⁺ transient and abolished subsequent events. During CPA exposure, a train of voltage steps to +10 mV induced I_Ca and transients of Ca²⁺ entry, but the caffeine response was not restored; however, upon CPA removal the Ca²⁺ transients were restored. A small transient increase of the resting [Ca²⁺]_i was induced by CPA, probably by inhibition of sarcoplasmic Ca²⁺ removal via the SERCA into the SR, which was subsequently dissipated by other Ca²⁺ removal mechanisms. Additional experiments showed that carbachol transients were also suppressed by superfusion with CPA. This result suggests that SERCA activity is required for Ca²⁺ to fill the intracellular Ca²⁺ store, and direct Ca²⁺ flux from the L-type Ca²⁺ channel to the SR is unlikely.

Figure 6. Cyclopiazonic acid prevents Ca²⁺ store refilling.

Caffeine-induced Ca²⁺ transients in a detrusor cell under voltage clamp. Cyclopiazonic acid (CPA; 20 μm) was added to the superfusate for the period indicated. The cell was depolarised by a train of pulses (-60 to +10 mV, 500 ms duration) between the last two caffeine exposures in CPA.

Possible mechanisms for activation of the L-type Ca²⁺ channel during refilling

The experiments above have implicated Ca²⁺ channels in mediating the Ca²⁺ influx that maintains intracellular Ca²⁺ stores. Because channel conductance is dependent on the membrane potential E_m, this was recorded during some of the conditions described above that affect the level of store filling. Figure 7A shows that brief exposure to a zero-Ca²⁺ solution reversibly reduced the [Ca²⁺]_i and simultaneously generated oscillating depolarisations. The depolarisations were also recorded using microelectrodes, which excludes possible artifacts in zero-Ca²⁺ solutions using patch electrodes either from dialysis of the intracellular contents or from a reduced stability of the patch seal. The E_m changes may have resulted from reduced screening of negative surface charges on the outer face of the membrane in the zero-Ca²⁺ solution. However, this was unlikely as superfusate CaCl₂ was replaced by equimolar addition of MgCl₂. Thus it is most likely that the membrane depolarisation occurs as a result of a reduced [Ca²⁺]_i.

Figure 7. The relationship between [Ca²⁺]_i and membrane potential.

A, simultaneous recordings of [Ca²⁺]_i and E_m in a detrusor cell under current clamp. The cell was exposed to a zero-Ca²⁺ solution for the period indicated. B, simultaneous recording of [Ca²⁺]_i and E_m during exposure to 20 mm caffeine.

Further evidence of a role for intracellular Ca²⁺ in determining E_m was sought during caffeine addition in a normal extracellular Ca²⁺ solution. In Fig. 7B the recording time was longer than in Fig. 1 to include the pronounced undershoot of [Ca²⁺]_i after the Ca²⁺ transient. The Ca²⁺ transient itself was associated with a brief hyperpolarisation but the subsequent undershoot of [Ca²⁺]_i was accompanied by a prolonged depolarisation, large enough (about 25 mV) to favour opening of L-type Ca²⁺ channels. To exclude specifically the chance that this effect of caffeine was due to an increase of cyclic nucleotides from its phosphodiesterase activity, similar experiments used another methylxanthine, IBMX (3-isobutyl-1-methylxanthine, 0.5 mm), that also has potent phosphodiesterase inhibitory effects. The characteristic changes to intracellular Ca²⁺ and membrane potential induced by caffeine could not be duplicated by IBMX, and excludes complications due to cyclic nucleotide accumulation.

To assess the importance of membrane potential in determining Ca²⁺ store refilling, the time course of the Ca²⁺ transient undershoot after caffeine undershoot was measured as a function of E_m. Figure 8 shows a paired experiment in which the time constant τ for recovery of the caffeine-induced Ca²⁺ undershoot was measured whilst the cell was clamped at either −60 or −25 mV. In eleven similar experiments τ was shortened from 118 ± 32 s at −60 mV to 69 ± 18 s at −25 mV (P < 0.05, n = 12). Similar results were obtained from a set of current-clamp experiments; τ was 105 ± 66 s at E_m −60 mV and decreased to 75 ± 15 s at E_m around −25 mV (P < 0.05, n = 11). These results show that recovery from the Ca²⁺ undershoot is voltage dependent and that depolarisation during this undershoot, following Ca²⁺ store depletion, plays a role in promoting recovery of resting [Ca²⁺]_i and Ca²⁺ store refilling.

Figure 8. Recovery of caffeine-induced Ca²⁺ transient undershoot at different membrane potentials.

Time course of recovery of [Ca²⁺]_i from the undershoot following caffeine-induced [Ca²⁺]_i release. Curves are the least-squares fit through the data points with a single exponential equation: A=A₀ (1 – exp(- (t–t₀)/τ)). The time course was measured at a holding potential of −60 mV and −25 mV under voltage clamp.

These results suggest that an electrophysiological event sensitive to changes of [Ca²⁺]_i could provide the link between E_m and [Ca²⁺]_i. One possibility is a Ca²⁺-activated K⁺ current that is present in detrusor muscle during normal activity (Klockner & Isenberg, 1985; Wu et al. 1999b). Control voltage-clamp experiments demonstrated spontaneous transient outward currents (STOCs) after generation of an inward Ca²⁺ current, which were abolished completely either by 100 nm iberiotoxin, the BK_Ca channel antagonist (Galvez et al. 1990), or superfusion with zero-Ca²⁺ Tyrode solution.

Evidence for the diminished involvement of a Ca²⁺-activated transient outward current during [Ca²⁺]_i reduction and store refilling was provided by the experiment shown in Fig. 9A. E_m and [Ca²⁺]_i were measured simultaneously during application of 20 mm caffeine; the cell was under current clamp with an E_m of −20 to −30 mV to enhance the transient hyperpolarisations mediated by Ca²⁺-activated K⁺ current. During the caffeine-induced Ca²⁺ transient, a single larger hyperpolarisation was recorded, followed during the undershoot of the Ca²⁺ transient by a much reduced activity of transient E_m changes. Similar results were observed if carbachol was used to elicit the Ca²⁺ transient.

Figure 9. [Ca²⁺]_i and spontaneous changes to membrane potential and membrane current.

A, simultaneous record of [Ca²⁺]_i and E_m in a detrusor cell during exposure to 20 mm caffeine. E_m was under current clamp at between −20 and −30 mV when the cell was quiescent before the recording period began. B, simultaneous record of [Ca²⁺]_i and membrane current in a detrusor cell during three applications of 20 mm caffeine.

Figure 9B shows that under voltage clamp, application of caffeine generated a large STOC during the Ca²⁺ transient itself, followed by complete cessation of activity during the Ca²⁺ transient undershoot until the [Ca²⁺]_i had returned to the pre-intervention level and the store had refilled. This suggests that intracellular Ca²⁺, and more importantly the [Ca²⁺]_i around the space between the sarcolemmal membrane and the peripheral SR and which is sensitive to the level of stored Ca²⁺ in the SR, regulates the BK_Ca channel and thus the average membrane potential. Such a hypothesis would predict a role for the BK_Ca channel in regulating the resting membrane potential in detrusor muscle. To test this E_m was measured during application of 100 nm iberiotoxin and a depolarisation of 16.6 ± 5.9 mV (n = 4) was recorded; E_m was recorded with microelectrodes, to minimise perturbation of the intracellular space.

Refilling assessed by muscle contraction measurements – effect of depolarisation and Ca²⁺ channel blockade

The above experiments using single isolated cells have important implications regarding the contractile behaviour of detrusor smooth muscle, as this is critically determined by the [Ca²⁺]_i, although quantitative extrapolation is difficult. To assess the functional relevance, tension experiments were performed using similar interventions to those used above. Figure 10A shows that brief application of a maximally effective carbarchol concentration (20 μm) produced reproducible contractile events in isolated detrusor muscle strips. Elevation of extracellular K⁺ from 4 to 16 mm did not itself produce significant contractions (except small contractile oscillations), but increased the subsequent carbachol contraction by 110 ± 2 % (P < 0.05, n = 11). Figure 10B shows that 10 μm nifedipine added in the interval between successive applications of carbachol decreased the following carbachol contraction. On average, carbachol-induced contractile force was reduced to 14 ± 2 % of control (P < 0.01, n = 6). These results are qualitatively consistent with the equivalent experiments measuring intracellular Ca²⁺ transients under these conditions (Fig. 4 and Fig. 5).

Figure 10. Effect of membrane depolarisation and L-type Ca²⁺ channel blockade on carbachol-induced contractions.

A, effect of depolarisation on maximal (20 μm) carbachol-elicited isometric contractions of a detrusor muscle strip; superfusate KCl was raised to from 4 to 16 mm between the second and third contractions. B, effect of an L-type Ca²⁺ channel antagonist; 10 μm nifedipine was added between the second and third contractions.

Refilling assessed by muscle contraction measurements – effect of CPA

The role of SERCA in Ca²⁺ store refilling was examined using carbachol-induced contractions induced 3–4 min after removal of extracellular Ca²⁺. Under these conditions the contraction was attributed mainly to intracellular Ca²⁺ store release and contributions from extracellular Ca²⁺ entry were minimised. Thinner strips (< 0.3 mm in diameter) were used and CPA was superfused for a long period (1.5–2 h) to maximise its penetration into the cells. A high extracellular [K⁺] (64 mm) was applied briefly prior to Ca²⁺ removal, and the contraction, which served as an internal control, was used as a measure of Ca²⁺ entry. The intervention was also a conditioning stimulation to enhance Ca²⁺ store refilling. Figure 11 shows that the carbachol-induced contraction in zero-Ca²⁺ was reduced after pre-incubation with 10 μm CPA, although the 64 mm K⁺-induced contraction was unaffected. The average carbachol contraction in zero-Ca²⁺ was reduced to 53 ± 10 % of control (P < 0.05, n = 10), whilst the 64 mm K⁺ contraction was 110 ± 7 % of control (P > 0.05). Thus SERCA inhibition reduced contractile force associated with Ca²⁺ released from intracellular stores, whilst the contraction due to extracellular Ca²⁺ entry was unaffected, and thus SERCA plays a functional role in Ca²⁺ store replenishment. A complete block of the carbachol contraction in zero-Ca²⁺ solution was difficult to achieve in the intact muscle strips, which may be due to a difficulty of CPA reaching the SR in sufficiently high concentrations throughout the preparation, despite their very small size.

Figure 11. Effect of CPA on carbachol contractions in zero-Ca²⁺ solution.

A contraction in normal Tyrode solution was elicited by application of 64 mm KCl followed by zero-Ca²⁺ solution during which 20 μm carbachol was applied. Left, control; right, pre-incubation with 10 μm CPA for 90 min.

DISCUSSION

L-type Ca²⁺ channels and cholinergic activation

Carbachol-induced Ca²⁺ transients result from intracellular Ca²⁺ release, as they could be elicited when the extracellular solution was about 10 nm (Wu & Fry, 1998). The intracellular source is likely to be the SR, as depletion of this store by prior treatment with caffeine abolished the subsequent carbachol response. The action of carbachol is likely to be mediated by IP₃ production, as the agonist increases inositol phosphate production over a similar concentration range that generates Ca²⁺ transients (Iacouvou et al. 1990) and the Ca²⁺ transient was abolished by prior treatment with TMB-8 (Fig. 1). The efficacy of IP₃-mediated release is high as the carbachol-induced Ca²⁺ transient has a similar magnitude to that of the caffeine transient and application of the maximum effective concentration of carbachol (20 μm) abolished the caffeine transient. Such a system would therefore function efficiently to provide activator Ca²⁺ for the contractile machinery, irrespective of any Ca²⁺ entry from the sarcolemma.

Detrusor myocytes also express L-type Ca²⁺ channels at a current density sufficient to support the action potential upstroke (Montgomery & Fry, 1992). However, several lines of evidence suggest that membrane depolarisation, and hence L-type Ca²⁺ channel activation, is unimportant in the genesis of the carbachol-induced Ca²⁺ transient: (i) the L-type Ca²⁺ channel antagonists verapamil and nifedipine had no significant effect; (ii) membrane potential changed very little during carbachol application, a small membrane depolarisation sometimes followed the peak of the Ca²⁺ transient and (iii) the Ca²⁺ transient magnitude was independent of membrane potential when clamped between −80 and +20 mV. The Ca²⁺ transient was often accompanied by a hyperpolarisation, reflecting Ca²⁺-dependent K⁺ current activation, which may play a role in limiting excessive Ca²⁺ entry and hence Ca²⁺ overload. The cause of the depolarisation that followed the Ca²⁺ transient was unclear, however; if large enough it could open L-type Ca²⁺ channels and cause intracellular Ca²⁺ oscillations (Kohda et al. 1996).

Refilling of functional intracellular Ca²⁺ stores

Using maximum concentrations of agonists, releasable Ca²⁺ was completely discharged from the SR into the sarcoplasm, and the functional Ca²⁺ pool was empty as there was no further response to an immediate second stimulus. The lack of effect of the second exposure was not due to receptor desensitisation, as an alternative agonist acting via a separate mechanism was equally ineffective in releasing Ca²⁺. Following agonist removal refilling commenced after a delay of about 15 s, estimated from extrapolation of the refilling curve in Fig. 2C. This absolute refractory period may represent the time taken for Ca²⁺ to be transported from the re-uptake to release site, or the time for the Ca²⁺ pool to accumulate to a threshold level for further release, as there is evidence that Ca²⁺ release is a function of the luminal [Ca²⁺] (Missiaen et al. 1992). Subsequently, the response recovered with an exponential time course and a time constant (τ) of about 90 s. This is similar to the time course of recovery of the undershoot of the caffeine-induced Ca²⁺ transients in these experiments (107 ± 12 s; mean ± s.e.m., n = 7) and also those reported in myocardium and rat hippocampal neurones (Baro et al. 1993; Garaschuk et al. 1997). Such an interval is sufficient for detrusor muscle which contracts only intermittently during voiding and suggests that a major contributory factor to recovery of the undershoot is SR store refilling. From an experimental viewpoint an interval between stimuli in refilling experiments of at least 5τ was used, which would achieve more than 98 % refilling.

Ca²⁺ store refilling is dependent on extracellular Ca²⁺, via Ni²⁺-sensitive routes. The store Ca²⁺ content is also determined by the resting [Ca²⁺]_i, as when this is low, e.g. in a zero-Ca²⁺ solution, the store cannot be refilled. Even in the absence of any triggered Ca²⁺ release, the Ca²⁺ store was depleted in the zero-Ca²⁺ solution. Refilling only commenced when the resting [Ca²⁺]_i began to rise, and full restoration of the Ca²⁺ pool occurred only after complete recovery of the resting [Ca²⁺]_i. These observations suggest that in detrusor cells the exchange of Ca²⁺ between the extracellular space, the sarcoplasmic pool and the SR lumen are all in dynamic equilibrium.

The role of the L-type Ca²⁺ channel in Ca²⁺ store refilling

One major component of the Ni²⁺-sensitive refilling pathway is the L-type Ca²⁺ channel, and evidence for involvement of this channel has come from several experiments using carbachol or caffeine to estimate the content of intracellular Ca²⁺ stores. (1) Membrane depolarisation enhanced store filling, after depletion by Ca²⁺ deprivation (Fig. 4). This was achieved either by raising the extracellular [K⁺] from 4 to 24 mm or under voltage clamp by changing E_m from −60 to −25 mV, when Ca²⁺ entry via the L-type Ca²⁺ channel window current should be maximal (Montgomery & Fry, 1992). (2) Enhancement was achieved if E_m was changed during the interval between successive applications of agonist under normal conditions of constant extracellular [Ca²⁺] (Fig. 4C). (3) Brief depolarising steps under voltage clamp, to elicit a Ca²⁺ current, enhanced refilling (Fig. 5C). (4) Ca²⁺ store refilling was attenuated by verapamil or nifedipine after store depletion by either extracellular Ca²⁺ removal or application of carbachol or caffeine in normal extracellular Ca²⁺ (Fig. 5), and (5) Enhancement of refilling by elevated extracellular [K⁺] and attenuation of the refilling by nifedipine, measured as carbachol-elicited muscle contraction, further supported the functional relevance of the above findings.

The small role of L-type Ca²⁺ channels in the carbachol-induced rise of [Ca²⁺]_i and their larger role in attenuating replenishment of functional Ca²⁺ stores explains both the smaller inhibitory effect of Ca²⁺ antagonists on carbachol-induced contractions compared with those generated by high K⁺ or ATP (Kishii et al. 1992), and also the progressively greater inhibition of carbachol-induced actions during continued exposure (Mostwin, 1985).

Refilling pathways

The experiments give insight into the processes that fill intracellular Ca²⁺ stores. It has been proposed that sarcolemmal Ca²⁺ entry could load preferentially superficial SR from a restricted sarcoplasmic micro-domain before it diffuses into the bulk sarcoplasm (Chen & van Breemen, 1993; van Breemen et al. 1995). Although a SERCA pump is required, store refilling would be independent of a change to the bulk sarcoplasmic [Ca²⁺]. Such a process is unlikely in detrusor muscle as depletion and replenishment of the Ca²⁺ store was affected by bulk resting [Ca²⁺]_i. Recovery of resting [Ca²⁺]_i occurred before restoration of the Ca²⁺ store pool (Fig. 3), which suggests that sarcolemmal Ca²⁺ influx enters the bulk sarcoplasm before substantial SR refilling.

In some non-excitable cells Ca²⁺ influx enters the SR via a direct low-resistance pathway, rather than uptake by a SERCA pump (Putney, 1986). Evidence for such a pathway was also provided in a rat aortic smooth muscle cell line (A7r5) by the observation that Mn²⁺ influx occurred without involvement of the SERCA pump, after SR depletion (Missiaen et al. 1990). In tracheal and oesophageal smooth muscle, a refilling pathway for the acetylcholine-sensitive Ca²⁺ store, which is sensitive to Ca²⁺ antagonists, is independent of the SERCA-specific inhibitor cyclopiazonic acid (CPA) (Bourreau et al. 1993; Liu & Farley, 1996; Salapatek et al. 1998). It has also been proposed that a preferred pathway exists whereby Ca²⁺ enters stores directly via L-type Ca²⁺ channels, bypassing the sarcoplasm (Janssen & Sims, 1993). In detrusor muscle, however, sarcoplasmic Ca²⁺ enters the SR mainly via the SERCA pump as CPA abolished subsequent Ca²⁺ release by caffeine (Fig. 6). Suppression by CPA of carbachol-elicited muscle contractions attributed to intracellular Ca²⁺ release was also qualitatively demonstrated (Fig. 11). Furthermore when Ca²⁺ entry was enhanced by depolarisation under voltage clamp, caffeine was still unable to generate a Ca²⁺ transient in the continued presence of CPA. Therefore, changes to SERCA pump activity significantly affect SR store refilling in detrusor muscle.

Although Ca²⁺ antagonists attenuated refilling of Ca²⁺ stores, they were unable to block it completely. Ca²⁺ entry via T-type Ca²⁺ channels has been described in detrusor (Sui et al. 2001), and in anococcygeous muscle store depletion-induced Ca²⁺ entry via these channels has been reported (Wayman et al. 1997). However, a major involvement of these channels in refilling is unlikely in detrusor muscle. Our experiments show that Ca²⁺ antagonists attenuated Ca²⁺ refilling to a similar degree in the absence or presence of 0.2 mm NiCl₂, a concentration that preferentially blocks the T-type Ca²⁺ channels (Sui et al. 2001). Moreover, the concentrations of Ca²⁺ antagonists used will not affect T-type channel conductance (Stengel et al. 1998). Other potential entry pathways that require further investigation are reverse mode Na⁺-Ca²⁺ exchange (Wu & Fry, 2001), non-specific cation channels or leak pathways as in a rat pheochromocytoma cell line (Bennett et al. 1998).

Mechanisms for the activation of the L-type Ca²⁺ channel during store depletion and refilling

Reduction of [Ca²⁺]_i, which occurred during zero-Ca²⁺ superfusion or after a caffeine Ca²⁺ transient, was accompanied by membrane depolarisation (Fig. 7). These changes were recorded with both microelectrodes and patch electrodes, so that the E_m changes were not an artifact of dialysis of the intracellular contents or stability of the patch seals. Such an effect has important implications as reduction of [Ca²⁺]_i will depolarise the membrane and consequently favour Ca²⁺ entry through L-type Ca²⁺ channels. Depolarisation during the undershoot following Ca²⁺ release may play a role in promoting store refilling as the time course of recovery from the undershoot was membrane potential dependent, and depolarisation facilitated the rate of recovery (Fig. 8). Conversely, Ca²⁺ influx would be reduced when the [Ca²⁺]_i is high. One mechanism for this phenomenon is a reduction of outward current through Ca²⁺-activated K⁺ (BK_Ca) channels as spontaneous transient outward currents (STOCs) were abolished during zero-Ca²⁺ superfusion or the undershoot of caffeine and carbachol Ca²⁺ transients. This would favour membrane depolarisation as they contribute substantially to net membrane conductance.

The particular importance of this current in mirroring changes to [Ca²⁺]_i and store refilling was evident in that the time courses for their recovery mirrored that for return of the [Ca²⁺]_i to normal after the Ca²⁺ transient and the restitution time of the Ca²⁺ transients themselves. The STOC magnitude and frequency is sensitive to the local [Ca²⁺]_i in the space immediately beneath the sarcolemma (Bolton & Imaizumi, 1996), and may be sensitive to the level of stored Ca²⁺, which can influence the release of Ca²⁺ into this sarcoplasmic space. The level of stored Ca²⁺ then could be represented by STOC activity. Consistent with this is the fact that a large STOC and membrane hyperpolarisation often preceded the peak of the Ca²⁺ transient, but that during the remainder of the transient STOC activity was greatly suppressed and a secondary membrane depolarisation occurred. This therefore suggests that intracellular Ca²⁺, particularly the [Ca²⁺]_i in the ‘fuzzy space’ immediately adjacent to the sarcolemma, is importantly involved in regulating the BK_Ca channel and in turn the membrane potential. We propose that [Ca²⁺]_i in the vicinity of the sarcolemma acts as a messenger for the functional SR content via Ca²⁺-activated K⁺ channels. Depletion of the store would lead to membrane depolarisation and Ca²⁺ entry via L-type Ca²⁺ channels.

In summary, the present study shows that the rise of intracellular [Ca²⁺] following activation of cholinergic receptors is independent of the membrane potential, but the opening of the L-type Ca²⁺ channels can be modulated by secondary effects on the membrane. The L-type Ca²⁺ channel plays an important role in refilling the functional intracellular Ca²⁺ stores, which would then influence normal detrusor contractile activity.