Smooth Muscle Group, Department of Physiology, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom
Submitted 6 September 2002 ; accepted in final form 25 March 2003
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ABSTRACT |
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interstitial cells; urethra; calcium-activated chloride current; calcium-induced calcium release; inositol 1,4,5-trisphosphate; ryanodine
The purpose of the present study was to distinguish between these possibilities by establishing whether 1) depolarization-induced Cl- current required influx of external Ca2+, and 2) intracellular stores are involved in depolarization-induced activation of the Cl- current.
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METHODS |
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Cell dispersal. Strips (0.5 cm) of proximal urethra were cut into
1-mm3 pieces and stored in Hanks' Ca2+-free
solution for 30 min before they were incubated in an enzyme medium containing
(per 5 ml of Hanks' Ca2+-free solution) 15 mg of
collagenase (Sigma type 1a), 1 mg of protease (Sigma type XXIV), 10 mg of BSA
(Sigma), and 10 mg of trypsin inhibitor (Sigma) for 5 min at 37°C.
They were then placed in Hanks' Ca2+-free solution and
stirred for a further 510 min to release both single relaxed smooth
muscle cells and IC. These were placed in petri dishes containing Hanks'
solution (100 µM Ca2+) and stored at 4°C for use
within 8 h.
Recordings were made using the amphotericin B perforated-patch method
(20). After gigaseals were
obtained, the series resistance fell over a 10- to 15-min period to
1015 M and remained stable for up to 1 h. Voltage-clamp commands
were delivered with an Axopatch 1D patch-clamp amplifier (Axon Instruments),
and currents were recorded by means of a 12-bit
analog-to-digital/digital-to-analog converter (Lab-master, Scientific
Solutions) interfaced to an Intel computer running pCLAMP software (Axon
Instruments). During experiments, the dish containing the cells was superfused
with bath solution (solution 2). In addition, the cell under study
was continuously superfused with bath solution by means of a close delivery
system consisting of a pipette (tip diameter 200 µm) placed
300 µm
away. This could be switched, with a dead space time of around 5 s, to a
solution containing a drug. All experiments were carried out at 37°C.
The solutions used were of the following composition (in mM): 1) Hanks' Ca2+ free solution (cell dispersal): 141 Na+, 5.8 K+, 130.3 Cl-, 15.5 HCO3 -, 0.34 HPO4 2-, 0.44 H2PO4 -, 10 dextrose, 2.9 sucrose, and 10 HEPES, pH adjusted to 7.4 with NaOH. 2) Bath solution: 130 Na+, 5.8 K+, 135 Cl-, 4.16 HCO3 -, 0.34 HPO4 2-, 0.44 H2PO4 -, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO2-4, 10 dextrose, 2.9 sucrose, and 10 HEPES, pH adjusted to 7.4 with NaOH. In some experiments, nominally Ca2+-free conditions were created by replacing the Ca2+ in this solution with equimolar Mg2+ and adding 5 mM EGTA. 3)Cs+ pipette solution: 133 Cs+, 1 Mg2+, 135 Cl-, 0.5 EGTA, and 10 HEPES, pH adjusted to 7.2 with CsOH.
The following drugs were used: caffeine (Sigma), ryanodine (Sigma), 2-aminoethoxydiphenyl borate (2-APB; Acros), cyclopiazonic acid (CPA; Calbiochem), xestospongin C (Calbiochem), and 2-nitro-4-carboxyl-N,N-diphenylcarbamate (NCDC; Sigma). Data are presented as means ± SE, and statistical differences were compared using Student's paired t-test, taking the P < 0.05 level as significant. n refers to the number of cells in series of experiments; a minimum of three animals was used for each data set.
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RESULTS |
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In the initial part of the present study, the requirement for external Ca2+ for evoking a Cl- tail current was investigated and characterized. In Fig. 1A, Ca2+ current (ICa) and Cl- tail current were evoked by stepping to -20 mV from a holding potential of -80 mV, followed by a step back to -80 mV while superfusing with normal (1.8 mM) Ca2+. External Ca2+ was then removed by switching the superfusate to nominally Ca2+-free 5 s before the next depolarizing step. This caused abolition of both ICa and the Cl- tail. This was confirmed in a total of four experiments in which the Cl- tails were abolished in Ca2+-free conditions (control tail -1,114 ± 226 pA, tail in Ca2+-free -23 ± 7 pA; n = 4; P < 0.01). In a second series of experiments, the effect of substituting Ba2+ for Ca2+ was investigated (Fig. 1B). In this case, the L-type Ca2+ current was enhanced, but the Cl- current was greatly reduced from -670 ± 153 to -104 ± 27 pA (n = 6; P < 0.01). Finally, the requirement for Ca2+ was examined by dialyzing the slow Ca2+ buffer EGTA into the cell. In these experiments, ICa and Cl- tail were evoked, first under perforated-patch recording conditions and then after the patch was ruptured by applying suction to the pipette (Fig. 1C). Pipette solution containing SO4 EGTA (2 mM) was allowed to dialyze into the cell for 20 s before the step to 0 mV was repeated. This abolished the Cl- tail current while having little effect on the amplitude of ICa. It is unlikely that the disappearance of the tail current was due to "run down," because, in our experience, run down of the Cl- current in whole cell recording occurs gradually over 510 min after rupture of the patch in parallel with run down of ICa.In these experiments, the current disappeared after only 20 s of dialysis after patch rupture and ICa was maintained at this time. In six cells, rupture of the patch and dialysis of 2 mM EGTA reduced the tail current from -688 ± 326 to -13 ± 4 pA (P < 0.05). We previously reported that nifedipine blocked the Cl- currents evoked by depolarization (22). Together with the present results, these data suggest that depolarization-induced activation of the Cl- channels is absolutely dependent on Ca2+ influx. It is unlikely, therefore, that voltage-dependent activation of the Cl- channels, voltage-dependent production of IP3 (17), or physical coupling of L-type Ca2+ channels to ryanodine receptors (c.f., skeletal muscle, Ref. 25) play any role in the activation of the Cl- channels in these cells.
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To test whether activation of the Cl- current also involved release of intracellular Ca2+, the effects of a variety of drugs that are known to interfere with intracellular Ca2+ stores were examined. Figure 2A shows the effect of CPA, an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. In these experiments, current-voltage (I-V) relationships were evoked by stepping protocols that involved holding the cell at -60 mV and then stepping to a series of potentials from -80 to +50 mV. In the control, this protocol evoked a series of inward Ca2+ currents that could be resolved on steps to -40 through to +30 mV (Fig. 2A). Slower activating Cl- currents could also be observed. These became fully activated after 100 to 300 ms and were inward at -40 to -10 mV and outward at +10 to +50 mV. On stepping down to -80 mV, Cl- tail currents could be observed. In the example shown in Fig. 2A, CPA reduced the Cl- currents without affecting ICa. [In these experiments, it was confirmed that the stores had emptied by showing that the caffeine-evoked Cl- current was abolished by CPA (data not shown).] Figure 2B shows an I-V plot for ICa that was obtained by measuring the peak ICa at the beginning of each step. Figure 2C shows I-V plots for the Cl- current, obtained by measuring the current at the end of each step, when most of the ICa had inactivated. These data confirm that CPA blocked a large component of Cl- current (P < 0.05; n = 4) without reducing the L-type Ca2+ current.
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In another series of experiments, the effect of a high concentration of ryanodine (30 µM) was determined. This concentration would normally be expected to block the ryanodine receptors (RYR), rather than to lock them open in a subconductance state (5, 23). Because ryanodine binds to the RYR in the open state, all cells in this series were subjected to repeated doses of caffeine in the presence of ryanodine until the caffeine-induced Cl- current completely disappeared (generally after four exposures to caffeine at 80-s intervals). A previously acquired I-V relationship in each of these cells was then compared with the I-V obtained in the presence of ryanodine. Figure 3A shows that the effect of ryanodine was to reduce, but not abolish, the Cl- currents. Summary data in Fig. 3C confirm that ryanodine reduced the Cl- currents (P < 0.05; n = 11; measured at the end of the 500-ms test pulses) despite the fact that there was a moderate enhancement of ICa (Fig. 3B; n = 11; P < 0.05).
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These results suggest that RYR contributed to the activation of the
Cl- current by depolarizing steps. The possibility that
IP3 receptors (IP3R) also contributed was considered.
Initially, this was tested with the IP3 receptor blocker
xestospongin C. An example of its effect is shown in
Fig. 4, where currents were
evoked by stepping to -20 mV and then to -80 mV. This protocol evoked
ICa at the beginning of the sweep, followed by a large
Cl- current that had, in this example, a biphasic pattern of
activation consisting of an early phase (beginning after 10 ms) and a
late phase (beginning after
150 ms). On repolarization to -80 mV, a large
tail current was recorded. Xestospongin C had little effect on
ICa in this experiment but blocked the late phase of the
Cl- current, as well as the tail. In six experiments, xestospongin
C reduced the Cl- tails from -1,695 ± 635 to -331 ±
121 pA (P < 0.05). However, xestospongin C also had a variable
effect on ICa. In two of the six experiments, there was no
effect on Ca2+ current, but in the other four there was
a variable degree of block. Overall, the mean effect was to reduce
ICa from -124 ± 15 to -72 ± 25 pA
(P < 0.05; n = 6). To further assess the involvement of
IP3R, the effect of 2APB was examined. We have previously found
this drug to be a good discriminator between RYR- and
IP3-R-mediated responses
(21).
Figure 5A shows a
typical example were 2-APB (100 µM) reduced the Cl- currents
without affecting ICa. Summary data in
Fig. 5B show that
2-APB had no effect on ICa throughout the voltage range
(n = 7), but it effectively reduced Cl- current in the
same cells (Fig. 5C;
P < 0.05; n = 7).
Figure 5D shows the
effect of 2-APB in combination with CPA on the inward Cl- currents
evoked by steps to -20 mV (n = 4), where it is clear that the
addition of 2-APB after CPA had no further effect. This suggests that CPA can
deplete the store that is sensitive to blockade with 2-APB. We also tested the
effect of a third drug, NCDC, a blocker of phospholipase C. NCDC (100 µM)
effectively reduced the Cl- tails (from -821 ± 227 to -352
± 193 pA; n = 5; P < 0.05) but also reduced L-type
Ca2+ current by a similar proportion (from -111 ±
37 to -31 ± 28 pA; n = 5; P < 0.02).
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DISCUSSION |
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The contribution of CICR to the activation of Cl- currents has been assessed in several smooth muscle types (3, 6, 9, 15, 18). In guinea pig trachea and rabbit coronary artery myocytes, the Cl- tail current was reduced by caffeine and CPA, whereas neither of these drugs significantly affected the tail current in the rat or rabbit portal vein (6, 18), suggesting that activation of Cl- currents by CICR varies between cell types. The role of RYR in the activation of Ca2+-activated Cl- current has been elegantly demonstrated in bladder myocytes where it was shown that a depolarization-induced Cl- tail current was activated by widespread CICR throughout the cell (3). Interestingly, in this preparation, the tail currents sometimes had a complex shape, reflecting the spread of a Ca2+ wave along the cell. Similarly, in the present study, Cl- currents in rabbit IC often had complex kinetics during control conditions (see Figs. 2, 3, 4, 5), but these became simpler in the presence of drugs that interfered with intracellular Ca2+ stores. It seems likely that the simpler kinetics mirrored only Ca2+ influx, whereas the complex kinetics recorded under control conditions depended both on Ca2+ influx and the spread of CICR throughout the cell.
In the present study, not only RYR but also IP3R were necessary for the activation of the Cl- currents as they were reduced by xestospongin C and 2-APB. The latter was first described as a specific blocker of the IP3R Ca2+ release channel in a variety of cell types by Maruyama and colleagues (16) and has since been widely used for this purpose. Despite some evidence in nonexcitable cells that 2-APB may also block store filling currents (19), we have shown that it is a good discriminator between IP3-sensitive and RYR-mediated responses in rabbit IC (21). Thus it abolished norepinephrine-evoked Cl- currents but had no effect on either caffeine-evoked Ca2+ release or on spontaneous transient outward currents (STOCs), both of which are believed to be mediated by RYR (14). The present results therefore support the involvement of IP3R in depolarization-induced activation of Cl- currents.
It is interesting to speculate how both RYR and IP3R could be involved in the activation of Cl- current by depolarization. Certainly, the requirement for both types of receptors for inducing global Ca2+ increases inside the cell has many precedents (1, 2, 5). For example, Boittin et al. (2) showed that in portal vein myocytes, norepinephrine-induced Ca2+ waves could be blocked by dialyzing either RYR- or IP3R-specific antibodies into the cell. They demonstrated that RYR and IP3R were colocalized and proposed a model involving sequential activation of the receptors, where IP3-induced Ca2+ release was amplified by a Ca2+ wave propagated by RYR (2). Similarly, amplification of IP3R-mediated purinergic responses by RYR was observed in murine colonic myocytes (1). Recently, the idea of cooperativity between the two receptor types has been extended to spontaneous (i.e., non-agonist evoked) Ca2+ events (5). Gordienko and Bolton (5) showed that ryanodine blocked both spontaneous Ca2+ sparks and Ca2+ waves, whereas 2-APB and xestospongin C blocked only the latter. Their interpretation was that RYR were responsible for initiating sparks, but both RYR and IP3R were necessary for propagation of the Ca2+ waves. Although the mechanism of this cooperativity was not fully elucidated, it was suggested that basal activity of phospholipase C (PLC) resulted in IP3-induced Ca2+ release in the microdomain of the RYR, thus sensitizing them to CICR. Such a mechanism may also operate in rabbit urethral IC, because large spontaneous Cl- currents were blocked by either ryanodine or blockers of IP3R/PLC, but small spontaneous transient outward currents (mediated by RYR) were blocked only by ryanodine (21). Applying these ideas to the present study, it is possible that basal activity of PLC elevates Ca2+ in the vicinity of the RYR, thus sensitizing them to CICR by the Ca2+ that comes in during depolarization.
In conclusion, we have demonstrated that the depolarization-induced Ca2+-activated Cl- current in rabbit urethral IC is enhanced by Ca2+ release from stores by a CICR mechanism that requires both RYR and IP3R. CICR may provide a mechanism for synchronizing pacemaker activity across a network of IC in the urethra. Urethral IC generate large STICs due to activation of Ca2+-activated Cl- currents (21, 22). In single IC, these currents can generate electrical slow waves that resemble the slow waves in whole tissue recorded with sharp microelectrodes (7, 8), whereas smooth muscle cells were electrically quiescent. This led us to propose that the IC act as pacemakers that drive the bulk smooth muscle (22). For this pacemaker system to work, it would be necessary for the activity of a sufficiently large group of pacemaker cells to be synchronized before they could generate enough current to drive the bulk smooth muscle cells lacking in the pacemaker mechanism. It is unlikely that sufficient synchronization could be achieved by spread of intercellular Ca2+ waves across the network, because their velocity is limited to <100 µm/s by the rate of Ca2+ diffusion (13). However, Ca2+-release could be coordinated across the network if it was coupled to depolarization by CICR. Indeed, in the gastrointestinal tract, where a similar pacemaking model has been proposed, it has been shown that depolarization recruits further "spontaneous transient depolarizations" (STD) to produce the regenerative components of electrical slow waves (10, 11, 24, 26).
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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