Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release

Sohag N. Saleh and Iain A. Greenwood

Department of Basic Medical Sciences, Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, London, United Kingdom

Submitted 9 August 2004 ; accepted in final form 2 September 2004


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 ABSTRACT
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The present study describes the first characterization of Ca2+-activated Cl currents (IClCa) in single smooth muscle cells from a murine vascular preparation (portal veins). IClCa was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of IClCa relied on Ca2+ entry through dihydropyridine-sensitive Ca2+ channels because IClCa was abolished by 1 µM nicardipine and enhanced by raising external Ca2+ concentration or by application of BAY K 8644. IClCa was characterized by the sensitivity to Cl channel blockers and the effect of altering the external anion on reversal potential. Activation of IClCa after membrane depolarization was dependent on Ca2+ release from intracellular stores. Thus the amplitude of IClCa was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 µM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.


A NUMBER OF LINES OF EVIDENCE implicate Ca2+-activated Cl currents (IClCa) in vascular excitability. Agents shown to block IClCa attenuate the amplitude of contractions produced by exogenously applied receptor agonists in functional studies on rat aorta, mesenteric artery, and pulmonary artery (7, 8, 35, 44). In addition, norepinepherine-induced contractions of rat aortic strips were augmented markedly by reduction of extracellular Cl concentration ([Cl]) to 8 mM (23). Furthermore, inhibition of the Cl transporters involved in the accumulation of Cl, either chemically (see, e.g., Ref. 1) or in the case of the Na+-K+-2Cl cotransporter (NKCC1) by gene ablation, reduced systolic blood pressure and decreased vascular contractility in response to stimulation of {alpha}-adrenoceptors (28). Also, the spontaneous rhythmicity of whole portal veins (PV) isolated from NKCC1–/– mice was significantly less than that of PV from wild-type mice.

These latter studies highlight the powerful nature of experiments with transgenic animals and the benefit of research using murine models. However, there is a paucity of information on IClCa in murine blood vessels. A previous study conducted at our laboratory (5) showed that sustained IClCa could be evoked in myocytes isolated from murine PV using pipette solutions of known free Ca2+ concentration ([Ca2+]). In addition, this cell type expressed CLCA1, a member of a family of putative Ca2+-activated Cl channel genes. The aim of the present study was to characterize transient IClCa evoked in murine PV smooth muscle cells under more physiological conditions. The data in the present study show that IClCa generated by promoting Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) exhibited biophysical properties similar to currents recorded in smooth muscle cells from other species. Moreover, our studies revealed that the amplitude of IClCa relied on VDCC activation but was not determined solely by Ca2+ influx. This highlighted a crucial dependence on Ca2+ released from intracellular Ca2+ stores. These observations represent the first major description of IClCa recorded in perforated patch conditions in a murine smooth muscle preparation. Some of the findings were presented previously in abstract form (36).


    MATERIALS AND METHODS
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Balb/c mice (6–8 wk old) were killed by cervical dislocation and exsanguination. PV were excised free from connective structures after ligation immediately proximal to the liver. Single smooth muscle cells were isolated by enzymatic digestion followed by mechanical liberation. Strips of PV were incubated in physiological salt solution (PSS; see Solutions and statistics for composition) containing 100 µM CaCl2 and protease XIV (3 mg/ml; Sigma) at 36°C for 5 min. After a 5-min period in PSS only, the tissue was incubated in PSS supplemented with 6 mg/ml collagenase type 1A (Calbiochem). The cell suspension was kept on ice for the duration of the experiment (6–8 h).

Electrophysiology. All currents were recorded with the whole cell configuration (either ruptured or perforated patch variants) of the voltage-clamp technique using a List LM-PC amplifier connected to pCLAMP 9 data acquisition and analysis package. Cells were held at –60 mV and VDCC were generated by 250-ms step depolarizations from various potentials between –50 mV and +70 mV. The voltage dependence of VDCC inactivation was determined by a two-pulse protocol involving a 1.5-s prepulse to potentials between –100 mV and +40 mV, followed by a test step (Vt) to +10 mV for 250 ms. Estimation of the reversal potential (Erev) of IClCa was determined by a two pulse protocol that involved a step to 0 mV for 200 ms to open ICa and then Vt between –100 mV and +60 mV for 500 ms. Erev was calculated by plotting the amplitude of the current 20 ms after stepping to Vt against the test potential. Changes in the junction potential were minimized by use of a 150 mM KCl agar bridge. Membrane potential recordings were performed in the perforated patch configuration using the current clamp plus command setting on the amplifier. Active responses were evoked by the injection of 100–300 pA for 10–30 ms.

Isometric tension recordings. PV were ligated in situ using 3-0 silk-braided suture thread (Pearsells Sutures, Somerset, UK) immediately proximal to the porta hepatis and distal to the anastomosis of splenogastric vein and mesenteric vein. Connective tissue and fat were removed by sharp dissection, and the tissue was placed in a 10-ml organ bath containing Krebs solution maintained at 37°C and gassed with 95% O2-5% CO2 at a resting tension of 0.1 g. Changes in isometric tension were recorded using a BIOPAC Systems force transducer and Acknowledge software (version 3.7). Similarly to a previous study (39), all veins were spontaneously active within minutes of arrangement and maintained rhythmicity for the duration of the experiment.

Solutions and statistics. The composition of the PSS used for cell dispersion was (in mM) 125 NaCl, 5.4 KCl, 15.4 NaHCO3, 0.33 Na2HPO4, 0.34 KH2PO4, 10 glucose, and 11 HEPES. The internal solution had the following composition (in mM): 126 CsCl, 10 HEPES, 5 EGTA, 5 Na2ATP, and 4 MgCl2, and pH was set to 7.2 with CsOH. For perforated patch experiments, this solution was supplemented with 300 µg/ml amphotericin B (Sigma). Using this compound to gain electrical access normally took ~10 min and access resistance (Raccess) was monitored by repetitive application of 5-mV hyperpolarizations from the holding potential of –60 mV. Experiments were initiated when Raccess had decreased to <40 M{Omega}.

In all voltage-clamp experiments, cells were bathed in a solution that contained (in mM): 126 NaCl, 10 HEPES, 11 glucose, 1.2 MgCl2, and 1.5 CaCl2, with pH set to 7.4 with NaOH. For the current-clamp experiments, cells were bathed in PSS containing 1.5 mM Ca2+, and the internal solution used was the same as above but contained 126 mM KCl instead of CsCl to maintain conditions as physiological as possible. The PSS for the functional experiments was (in mM) 125 NaCl, 4.6 KCl, 15.4 NaHCO3, 1 Na2HPO4, 0.6 MgSO4, and 10 glucose. NFA, anthracene-9-carboxylate (A-9-C), nifedipine, BAY K 8644, tetracaine, and amphotericin B were purchased from Sigma UK. The SR-ATPase inhibitor cyclopiazonic acid (CPA) as well as 2-aminoethoxydiphenyl borate (2-APB) were obtained from Calbiochem. All reagents were dissolved in DMSO or distilled H2O, and equivalent volumes of solvents did not affect the conductances studied. All data are means ± SE of n cells from at least three different mice. Significance values were calculated using paired t-tests or ANOVA.


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Initial characterization of Ca2+ currents evoked by membrane depolarization. To allow VDCC to be studied in isolation, currents were recorded using the ruptured patch whole cell configuration with a pipette solution that contained Cs and EGTA. The former would remove any voltage-dependent K+ currents, while the latter agent buffered any rise in global [Ca2+] sufficient to prohibit the activation of Ca2+-dependent conductances. Under these conditions, a rapidly activating inward current with slow decay kinetics developed at potentials positive to –30 mV that reached a peak at 0 mV of 28 ± 3 pA (n = 20; Fig. 1A). Increasing extracellular [Ca2+] to 10 mM augmented the amplitude of the inward current (peak amplitude was 66 ± 7 pA). Further current enhancement was observed upon the application of 1 µM BAY K 8644 (peak amplitude was 100 ± 12 pA; n = 11). In comparison, replacement of extracellular Na+ with Tris had no effect on the evoked current (n = 4). Consistent with this current being due to the activation of L-type VDCC, application of 1 µM nicardipine abrogated the inward current (n = 7; Fig 1A). In comparison, the T-type Ca2+ channel blocker mibefradil had no effect on the inward current (data not shown; n = 4). Figure 1B shows the voltage dependence of activation and inactivation determined by fitting the normalized data generated by the respective protocols to a Boltzmann function of the following form:

This yielded values for half-maximal activation and inactivation (V0.5) in 1.5 mM Ca2+ of –9 ± 2 mV and –25 ± 1 mV, respectively (n = 13). Slope values (–K) were 7 ± 0.6 mV and 5 ± 1 mV. There was no significant effect of raising extracellular [Ca2+] on the voltage dependence of inactivation (mean V0.5 in 10 mM Ca2+ was –25 ± 3 mV; n = 14). These data show that depolarization of murine PV myocytes evoked an inward current with properties similar to those of L-type Ca2+ currents recorded previously in other nonmurine smooth muscle cells.



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Fig. 1. Properties of the Ca2+ current (ICa). A: current-voltage relationship of the peak inward current evoked in a pipette solution containing 5 mM EGTA and external solutions containing 1.5 mM Ca2+ (open triangles), 10 mM Ca2+ (closed squares), 10 mM Ca2+ and 1 µM BAY K 8644 (open circles), and 10 mM Ca2+ and 1 µM nicardipine (closed diamonds). B: activation and inactivation properties of the channel were measured using a double-pulse protocol in which cells were stepped from –60 mV to a range of voltages for 1.5 s, followed by a single 250-ms step to +10 mV. Inactivation (closed squares) values were normalized, and mean data were fitted with a Boltzmann function, giving a V0.5= –25 ± 1 mV, while activation was measured as normalized conductance value, giving a V0.5= –9 ± 2 mV. Data are means ± SE.

 
IClCa is generated under conditions of low buffering of intracellular [Ca2+]. When the perforated patch recording configuration was used, step depolarization elicited IClCa as a consequence of Ca2+ influx through VDCC in 131 of 160 cells in which ICa was evoked. This is highlighted in Fig. 2, which shows that the rapidly activating inward ICa evoked at +20 mV was superimposed by a slowly developing outward current, followed by an inward current that declined in an exponential manner (termed ITAIL) upon repolarization to –60 mV. In 24 cells, spontaneous inward currents at –60 mV were also recorded. These had a mean amplitude of 34 ± 2 pA, a slow rise time (mean 10–90% was 41 ± 2 ms), and decayed exponentially [mean time constant ({tau}) was 89 ± 2.9 ms]. These currents were identical to the spontaneous Cl currents recorded in other smooth muscle cells (17, 19, 43) and were not analyzed further in this study.



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Fig. 2. Dependence of an inward current that declined in an exponential manner (ITAIL) on Ca2+ influx. Representative traces showing currents evoked in the perforated patch configuration upon depolarization from Vh to +20 mV (inset) slowly developing outward current (ILATE). A: different currents (ICa, ILATE, and ITAIL) evoked in the perforated patch configuration using the protocol shown and their abolishment by nicardipine (1 µM). B: increasing the extracellular Ca2+ concentration ([Ca2+]) incrementally resulted in both ICa ({blacksquare}) and ITAIL ({circ}) amplitude increasing in a sigmoid manner. The EC50 value was ~3 mM for both currents. The inset shows the currents evoked at 1.5 and 10 mM external [Ca2+]. C: BAY K 8644 also augmented the currents, while membrane rupture with a pipette solution containing 5 mM EGTA (D) abolished ITAIL without an effect on ICa.

 
The depolarization-evoked currents were qualitatively similar to IClCa recorded in rabbit PV myocytes (12) as well as other smooth muscle cell types (see, e.g., Refs. 2, 6, 20, 41). Therefore, we undertook a number of experiments to confirm that in murine PV myocytes, IClCa were activated after Ca2+ influx. Reliance on Ca2+ influx through L-type VDCC was corroborated by the use of nicardipine (1 µM), which abolished ICa and ITAIL completely (n = 4; Fig 2A). Conversely, the amplitude of ITAIL was increased when ICa activation was augmented either by raising the external Ca2+ to 10 mM or by applying the Ca2+ channel activator BAY K 8644 (Fig. 2, B and C). These maneuvers increased peak ITAIL from –92 ± 13 pA (n = 21) to –182 ± 37 pA and –306 ± 86 pA, respectively (n = 21 and 6). The inability to record ITAIL in conventional whole cell mode with a pipette solution containing 5 mM EGTA reflects the Ca2+ dependence of this conductance. Similarly, ITAIL recorded in the perforated patch configuration was rapidly abolished if the membrane was ruptured by the application of a brief pulse of negative pressure to allow ingress of EGTA into the cell (Fig. 2D). ICa was not significantly affected by this maneuver.

Anion replacement experiments were used to determine the ionic nature of the currents activated as a consequence of Ca2+ influx through ICa. With the use of a two-pulse protocol, the Erev of ITAIL under control conditions was –1 ± 3 mV (n = 8), and this changed to –46 ± 4 mV and +17 ± 5 mV (n = 6 and 4) after replacement of the external NaCl by either NaSCN or Na-isethionate, respectively (Fig. 3A). These shifts in reversal potential were equivalent to a relative permeability for SCN and isethionate of 6.5 ± 0.8 and 0.3 ± 0.1 (n = 6 and 4), respectively. Changing the external anion also influenced the kinetics of ITAIL. With an external solution containing NaCl, ITAIL declined with a mean time constant ({tau}) of 86 ± 15 ms at –60 mV (n = 65). This decay became faster at more hyperpolarized potentials ({tau} at –100 mV was 66 ± 9 ms; n = 6). Bathing the cells in a solution containing 126 mM NaSCN slowed the decay of ITAIL at every potential (mean {tau} at –100 mV was 117 ± 15 ms; n = 6). These data are consistent with previous studies in rabbit PV and pulmonary artery (13, 32).



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Fig. 3. The effects of anion replacement and Cl channel blockers on ITAIL. A: series of representative traces generated in the perforated patch configuration in 10 mM Ca2+ external solution. The cell was depolarized from Vh to 20 mV for 250 ms to allow Ca2+ influx, and this was followed by a 500-ms step between –60 and 100 mV at 20-mV increments to establish an estimate of the reversal potential (Erev) of ITAIL (closed square) in 126 mM NaCl extracellular solution (a), 126 mM NaSCN (b), and 126 mM Na-isethionate (c). B: effect of the Cl channel blocker niflumic acid (NFA; 20 µM) on a current evoked by step depolarization to 20 mV showing a significant effect on ITAIL and no appreciable effect on ICa. C: the voltage-dependent Cl channel blocker anthracene-9-carboxylate (A-9-C) selectively inhibited the outward current during the test step with no significant effect on either ICa or ITAIL. For mean values, see IClCa is generated under conditions of low buffering of intracellular [Ca2+].

 
Further pharmacological studies were undertaken to confirm the identity of ITAIL. Niflumic acid (NFA) is considered the most potent blocker of IClCa in smooth muscle cells, although this agent is far from specific (see, e.g., Ref. 11). Application of 10 and 20 µM NFA inhibited ITAIL by 55 ± 8% and 74 ± 9% (n = 5), respectively. This was associated with marked slowing of the decay of ITAIL (mean {tau} at –60 mV in 20 µM NFA was 314 ± 60 ms). Application of 50 µM NFA inhibited ITAIL completely but also depressed ICa (~50%) in all cells studied. Pharmacological studies were also undertaken with A-9-C, an agent that is a markedly more potent blocker of IClCa at positive potentials than negative ones (see Refs. 17, 32). As Fig. 3C shows, 500 µM A-9-C reduced the outward IClCa recorded during the test step to +20 mV (mean inhibition was 42 ± 6%; n = 5). Surprisingly, A-9-C had no effect on ITAIL at –60 mV (mean effect 3 ± 1.5%). Overall, these data confirm that in murine PV myocytes, IClCa can be elicited as a consequence of Ca2+ influx through voltage-gated Ca2+ channels.

Dependence on Ca2+ influx for activation of IClCa. The data from the previous section show unequivocally that the generation of IClCa was dependent on Ca2+ influx through L-type VDCC. However, there was a considerable variation in the amplitude of ICa required to generate IClCa. Figure 4A shows examples of ICa and IClCa evoked by a step depolarization to +20 mV in different cells. In the cells shown in Fig. 4, Aa and Ac, ICa of ~100 pA generated an ITAIL of approximately –650 pA. In the cell shown in Fig 4Ab, an ICa of the same amplitude elicited a considerably smaller ITAIL (–260 pA). This poor correlation between the amplitude of ICa and the concomitant activation of ITAIL in an external solution containing 1.5 mM Ca2+ is illustrated in Fig. 4B. However, the relationship between the amplitude of ICa and that of ITAIL became stronger as the degree of Ca2+ influx was augmented, either by raising external Ca2+ or by the application of BAY K 8644 (Fig 4C). Moreover, there was a good correlation between the kinetics of ITAIL and the degree of activation, regardless of the external [Ca2+]. Figure 5 shows the {tau} value for the decay of ITAIL at –60 mV plotted against the amplitude of ITAIL, and it is clear that there is a good agreement (r = 0.79) between ITAIL amplitude and the concomitant decay. Together these data suggest that while the activation of IClCa is triggered by Ca2+ influx, other processes govern the ultimate activation of this current.



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Fig. 4. The relationship between ICa amplitude and ITAIL amplitude. A: traces from 3 different cells evoked using the protocol shown in identical intracellular and extracellular conditions (see Solutions and statistics). These traces highlight the inconsistent relationship between ICa and ITAIL (measured at points indicated). ICa amplitude was plotted against ITAIL amplitude in external solutions containing 1.5 mM Ca2+ (B), 10 mM Ca2+ (C, closed squares) and 1.5 mM Ca2+ + 1 µM BAY K 8644 (C, open stars). Data were fitted using linear regression, and the correlation coefficients were 0.29, 0.64, and 0.76 for 1.5 mM Ca2+, 10 mM Ca2+, and BAY K 8644, respectively.

 


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Fig. 5. Dependence of the decay kinetics of Ca2+-activated Cl currents (IClCa) on current amplitude. A: representative trace produced by the protocol shown with the {tau} values for decay of ITAIL indicated. B: the values for ITAIL amplitude (closed square) and corresponding ITAIL decay were plotted (n = 74), and the values were fitted by a linear function, giving a correlation value of r = 0.74.

 
A recent exhaustive study of pacemaker cells from rabbit urethra revealed that the amplitude of IClCa evoked as a consequence of Ca2+ influx was due in part to the release of Ca2+ from different intracellular Ca2+ stores (18). We therefore investigated the possibility that Ca2+ release from the sarcoplasmic reticulum (SR) prompted by nicardipine-sensitive Ca2+ influx contributed to the activation of IClCa in PV myocytes. Experiments were performed in cells bathed in 10 mM Ca2+, and the contribution of intracellular Ca2+ release was probed with a number of different agents. An initial contribution from intracellular stores was determined using CPA (42). Application of 10 µM CPA for 5 min produced a marked diminution of ITAIL recorded at –60 mV, with a mean inhibition of 60 ± 11% (n = 4, see Fig. 6A). This effect was slowly reversible after washout. Smooth muscle cells are known to have heterogeneous Ca2+ stores that are depleted either by the membrane lipid hydrolysis product, inositol 1,4,5-trisphosphate (IP3), or through Ca2+-induced release. CPA does not discriminate between either store. Consequently, we used pharmacological agents that modulate differentially the IP3-dependent and IP3-independent release channels (conventionally defined as ryanodine receptors). 2-Aminoethoxydiphenyl borate (2-APB) is a membrane-permeable inhibitor of IP3 release channels (26) that has good selectivity for these receptors over ryanodine receptor channels (see Refs. 18, 37). Application of 100 µM 2-APB rapidly decreased the amplitude of ITAIL (Fig. 6B), with a mean effect of 48 ± 11% (P < 0.01 n = 13). In 5 of 13 cells, 2-APB also inhibited ICa, but the overall effect (10 ± 8%) did not reach significance. An involvement of ryanodine receptor-mediated Ca2+ release was investigated with tetracaine, a cell-permeable agent that rapidly blocks the Ca2+ release channel (16, 30). In the presence of 50 µM tetracaine, the amplitude of ITAIL was markedly smaller compared with control conditions (Fig. 6C) (mean decrease was 56 ± 5%; n = 8), and this effect was readily reversed within 2 min of tetracaine washout. Application of tetracaine and 2-APB together produced significantly greater inhibition of IClCa than either agent applied alone (mean inhibition of ITAIL was 76 ± 6%; n = 4).



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Fig. 6. Effect of store modulators on ITAIL amplitude. A: representative trace showing the effect of the SR-ATPase inhibitor cyclopiazonic acid (CPA; 10 µM) on a current evoked by depolarization to +20 mV in 10 mM Ca2+ external solution. B: another trace from a different cell evoked in identical conditions, further highlighting the variability of the relationship between ICa and ITAIL and showing the effect of the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB; 100 µM). C: ryanodine receptor antagonist tetracaine (50 µM) had a similar inhibitory effect on ITAIL.

 
In the majority of cells, the effect of 2-APB and tetracaine on IClCa was not associated with an effect on ICa, but there was a small inhibition in some cells. While this effect was not significant (total effect was 13 ± 5%), it could be argued that any depression of IClCa could be due to decreased Ca2+ influx. We therefore undertook a series of experiments that circumvented any possible effect due to depression of ICa by raising external [Ca2+] from 1.5 to 10 mM in the absence and presence of the store modulators. Because the amplitude of ICa was augmented by this maneuver, any inhibitory effect of the store modulators on ICa would be obviated.

Figure 7 shows the time course for the increase in ITAIL generated by an increase in external [Ca2+] to 10 mM in the absence and presence of 2-APB, tetracaine and CPA. Under control conditions, ITAIL increased in a sigmoid manner with a time to reach half-maximal amplitude of 61 ± 5 s at a flow rate of 2 ml/min (n = 14). Restoration of the control external solution (1.5 mM [Ca2+]) led to a rapid reduction in IClCa amplitude. When the substitution of external solution occurred in the presence of 100 µM 2-APB there was an abrupt attenuation of the current development so that after 100 s, the increase in ITAIL was only 59 ± 13% compared with current developed in the absence of 2-APB (Fig. 7A; P < 0.05). After 160-s incubation in 10 mM Ca2+ plus 2-APB, ITAIL was only 13 ± 15% of control values (P < 0.01) even though the amplitude of ICa was considerably enhanced. Figure 7, B and C, shows that a qualitatively similar effect was observed with 50 µM tetracaine and CPA. In both cases, the increase in external Ca2+ from 1.5 to 10 mM failed to produce a marked increase in ITAIL even though there was a marked increase in ICa. The effects of tetracaine on the development of ITAIL ranged from a relatively small (30%) inhibition to >100% block, i.e., the ITAIL recorded in 10 mM Ca2+ and tetracaine was smaller than that recorded in 1.5 mM Ca2+ alone. Overall, these data show that Ca2+ release from IP3-dependent and IP3-independent Ca2+ stores triggered by Ca2+ influx through L-type VDCC contributes to the rise in [Ca2+] in the vicinity of the Cl channel, leading to generation of IClCa.



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Fig. 7. Effect of store modulators on ITAIL amplitude under conditions of enhanced Ca2+ influx. A: increasing the external [Ca2+] from 1.5 to 10 mM augmented the amplitude of ITAIL in a time-dependent manner. a: Under control conditions, the current reached maximal amplitude in a sigmoid manner within 180 s (closed squares), and this effect was reversible (Aa1 and Ab1). Repeating the protocol with 2-APB (100 µM, n = 5) added to the 10 mM Ca2+-containing external solution resulted in an initial increase in ITAIL amplitude (Aa2 and Ab2), followed by almost complete abolition of the increase within 180 s (Aa3 and Ab3). Typical traces from the time points indicated in Aa are shown in Ab. CPA (10 µM, n = 5; B) and tetracaine (50 µM, n = 5; C) were found to have a similar effect, although the effect of tetracaine was far more variable. Significance values were calculated using paired t-tests. *P < 0.05. **P < 0.01. ***P < 0.001.

 
Functional contribution of IClCa in murine PV. Our data in the previous sections show clearly that robust IClCa are present in murine PV myocytes. Therefore, we attempted to define a possible functional role for this conductance in PV rhythmicity. Current-clamp studies were undertaken in quasiphysiological conditions using the perforated patch configuration and K+-containing internal and external solutions. Under these conditions, the resting membrane potential was –44 ± 1.0 mV (n = 7) and injection of depolarizing current elicited a rapid potential deflection of 69 ± 2.5 mV that was followed by a slower and more persistent depolarization of 14 ± 2 mV (see Fig. 8A). Application of NFA reduced the afterdepolarization to 3.7 ± 0.6 mV (P = 0.0001) but did not affect the amplitude of the action potential (mean amplitude was 65 ± 3.7 mV; P = 0.15).



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Fig. 8. Functional contribution of IClCa in murine portal veins (PV). A: 2 examples of membrane depolarizations elicited by a 20-ms injection of current to single PV myocytes held in current clamp in the absence and presence of 10 µM NFA. Evoked responses were biphasic, consisting of a rapid initial depolarization and a more delayed plateau phase. The lag phase between the peak amplitude of the initial and late depolarizations varied between cells. B: isometric tension recording from the whole murine PV showing the effect of external Ca2+ removal on the spontaneous contractions; the effect was reversible and highly reproducible (n = 8). C: a similar recording showing the effect of NFA (10 and 30 µM) on murine PV rhythmicity (representative of 3 experiments). D: the lack of effect of NFA (30 µM) on a KCl-induced spasm (bottom trace) with a plot of the mean effect on tension against time (n = 3).

 
In isometric tension experiments, whole murine PV were spontaneously active. The activity was abolished by the application of nicardipine (1 µM) or by bathing the tissue in Ca2+-free solution (Fig. 8B). Application of 10 and 30 µM NFA also inhibited the spontaneous activity. This manifested as a decrease in the contractile frequency by 18 ± 9 and 60 ± 4% and a reduction in the amplitude of individual contractions by 21 ± 17 and 52 ± 15% produced by 10 and 30 µM NFA, respectively (n = 3). These effects were reversed readily upon washout. These concentrations of NFA had no significant effect on the contraction produced by the application of 60 mM KCl (see Fig. 8D). A final series of experiments revealed that 30 µM 2-APB inhibited spontaneous contractions, with a mean decrease in frequency from 13 ± 2 min–1 to 2 ± 0.6 min–1 (n = 3).


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The present study represents the first in-depth characterization of IClCa elicited in murine vascular smooth muscle under the quasiphysiological conditions afforded by the perforated patch technique. Our data show that IClCa in murine PV share a number of biophysical properties with currents evoked using the same technique in nonmurine cells. We also provide evidence that IClCa evoked as a consequence of membrane depolarization depended extensively on Ca2+ release from both IP3-dependent and IP3-independent intracellular Ca2+ stores. Finally, we have shown that IClCa contributed to the inherent rhythmicity of whole PV.

Characteristics of IClCa in murine PV myocytes. IClCa have been elicited after membrane depolarization in a number of different cell types. Of particular relevance to the present study are the characteristics of these currents in rat and rabbit PV smooth muscle cells (12, 31). In addition, similar currents have been recorded in rabbit esophagus (2), rabbit pulmonary artery (44), canine trachea (20), rabbit coronary artery (24), sheep lymph node (41), and sheep urethral smooth muscle (6). In all cases, the currents were evoked with conditions of low intracellular buffering of Ca2+ (perforated patch or 0.1 mM EGTA) and were inhibited by dihydropyridines and by Cl channel blockers. In the majority of studies, the evoked IClCa had complex kinetics at negative potentials, except in rabbit coronary artery and PV myocytes, in which the current decay conformed to a single exponential value (12, 24).

Investigators at our laboratory (5) previously showed that pipette solutions containing a known free [Ca2+] evoked sustained IClCa in murine PV myocytes. This technique is useful for studying putative modulators of IClCa because the channel is stimulated without any reliance on other proteins (see, e.g., Refs. 5, 14, 15, 25). However, the recording conditions do not faithfully represent a physiological scenario, in which the stimulation of IClCa is likely to be more transient. Therefore, the present report describes the first study to characterize IClCa in a murine cell type under relatively physiological conditions.

IClCa were recorded using the perforated patch to minimize any disturbance of the intracellular compartment and were evoked by a transient rise in [Ca2+] generated by increasing the open probability of VDCC. IClCa elicited under these conditions exhibited a number of characteristics.

First, the inward currents at negative potentials (ITAIL) decayed with a time course that was voltage dependent and fitted well by a single exponential value similar to ITAIL recorded in rabbit PV myocytes (12) as well as coronary artery (24). The decay of ITAIL was prolonged by substitution of the external Cl with the more permeable anion thiocyanate, similarly to ITAIL in rabbit PV (13). In contrast to ITAIL in rabbit PV myocytes, the decay time constant of ITAIL at –60 mV was related to the amplitude of the evoked Cl current.

Second, the relative permeability of thiocyanate (~7) and isethionate (~0.3) was consistent with prior reports of IClCa in other cell types (13, 14). Investigators at our laboratory (5) determined an identical relative permeability for the same anions in a previous study in which sustained IClCa were evoked by the free [Ca2+] in the pipette solution. Consequently, the biophysical factors that determine ion selectivity are not affected by the manner in which the channel is stimulated. This is compared with the efficacy of various agents to block IClCa that is crucially dependent on how the channel is stimulated (see Refs. 32, 33).

Third, similar to other studies (6, 24, 41), IClCa in the present study was inhibited by the Cl channel blockers NFA and A-9-C. Inhibition of IClCa at –60 mV was associated with a lengthening of the decay, whereas the voltage-dependent agent A-9-C inhibited IClCa at only positive potentials. Overall, our data show that IClCa in murine PV myocytes exhibited characteristics common to IClCa in other vascular myocytes. Because IClCa in murine PV smooth muscle cells do not appear to exhibit any unique properties, future experiments designed to study IClCa in transgenic animals are viable. It is also worth noting that qualitatively similar IClCa were recorded in the present study using a conventional, ruptured patch whole cell configuration with a pipette solution containing 0.1 mM EGTA. These data challenge the accepted dogma that the perforated patch recording mode is essential for recording IClCa.

Intracellular Ca2+ stores contribute to the activation of IClCa. Generation of IClCa in murine PV myocytes had an obligatory requirement for Ca2+ influx through VDCC that were biophysically identical to L-type VDCC recorded previously in vascular smooth muscle cells (see, e.g., Refs. 27, 29, 38). However, there was a distinct lack of a correlation between the size of ICa and the size of IClCa subsequently evoked, although increasing the degree of Ca2+ influx by raising extracellular [Ca2+] or applying BAY K 8644 improved the correlation between ICa and IClCa. These observations suggest that activation of IClCa after membrane depolarization involved some mechanisms in addition to simple Ca2+ influx.

In terms of regulation by intracellular signaling molecules, only the Ca2+-dependent phosphatase calcineurin has been shown to enhance the activation of IClCa in vascular smooth muscle cells (15, 25). However, a number of studies of IClCa have implicated an involvement of Ca2+-induced Ca2+ release. Consequently, our attention focused on the possible involvement of Ca2+ release from intracellular stores amplifying Ca2+ influx through VDCC. Because the molecular identity of the channel underlying IClCa is unknown, we were unable to perform any immunocytochemical studies to investigate how intracellular Ca2+ release channels localized with the Cl channel. Consequently, we relied on selective blockers of the SR-ATPase (CPA) and the two known intracellular release pathways, namely, the IP3 receptor and the ryanodine receptor. CPA inhibited ITAIL by ~70%, consistent with voltage-dependent Ca2+ influx promoting the release of Ca2+ from an intracellular pool in murine PV myocytes. 2-APB blocks the IP3 receptor (26) and has been shown to have good selectivity for this receptor over the ryanodine receptor in a number of studies (see, e.g., Refs. 18, 37). Tetracaine, in addition to blocking voltage-gated Na+ channels, is a selective blocker of ryanodine receptor-mediated Ca2+ release channels (16, 30). 2-APB and tetracaine applied alone inhibited ITAIL by ~40%, which suggests that Ca2+ release via IP3-dependent and IP3-independent channels contributed to the activation of IClCa. Ca2+-induced Ca2+ release has been shown to contribute to IClCa in guinea pig trachea, rabbit coronary artery, and interstitial cells from the rabbit urethra (18, 20, 24). Interestingly, in rat and rabbit PV myocytes, IClCa evoked by membrane depolarization were not affected by caffeine application (31) or incubation in CPA or thapsigargin (10). However, in rat PV myocytes, brief (<20 ms) depolarizations to +10 mV generated localized Ca inhomogeneities (imaged as Ca2+ "sparks") and Ca2+ waves that spread throughout the cell were elicited by longer (500 ms) depolarizations (3). Ryanodine or caffeine abrogated both types of Ca2+ increment but did not affect the global rise in [Ca2+] in rat PV myocytes as indicated by fura-2 fluorescence (21). These observations suggest that Ca2+ influx in PV myocytes can provoke release of Ca2+ from intracellular stores but that the ability of this Ca2+ increment to stimulate the Ca2+-dependent Cl channel is governed by other factors.

Previous studies of rat and rabbit PV myocytes have revealed that IP3 receptors and ryanodine receptors are colocalized (4, 9) and that both release channels contribute to the formation of Ca2+ waves. It has been suggested that, in rabbit PV myocytes and urethral interstitial cells, the elementary Ca2+ event is governed by Ca2+ release from ryanodine receptors and IP3 receptors are necessary to generate larger Ca2+ events. Our studies do not reveal whether both release channels are present in a common intracellular Ca2+ pool or whether each acts in parallel or in series. However, the two agents added together produced significantly greater depression of IClCa. This suggests that to some extent that Ca2+ release from the two channel types must occur contemporaneously and that release of Ca2+ mediated by one receptor does not deplete the store used by the other release process. Moreover, this argues for each agent acting only on its targeted release channel. Overall, these data suggest a complicated and dynamic subplasmalemmal scenario in which Ca2+ influx through VDCC raises [Ca2+] indirectly in the proximity of the Cl channel to activate IClCa. The mechanisms that govern this amplification are variable and complex.

While in the majority of cells 2-APB and tetracaine had no effect on ICa, some inhibition was observed in a number of cells, although the degree of inhibition did not reach significance. It is axiomatic that if ICa is required for activation of IClCa, then any decrease in this conductance may affect IClCa. Consequently, we undertook measures to ensure that the observed depression of IClCa was not due to reduced influx through VDCC. In these experiments, the external Ca2+ was increased to 10 mM to enhance Ca2+ flux, and the effect on IClCa was monitored. In the absence of any store modifiers, this manipulation augmented IClCa by ~100%. However, when the cell was bathed in PSS containing 10 mM Ca2+ and 2-APB, the increase in IClCa was considerably less (~20%). The same was observed for tetracaine and CPA. These data highlight the crucial role of Ca2+ release from intracellular stores in the activation of IClCa after membrane depolarization.

Role of IClCa in PV rhythmicity. Contraction of the whole PV is coordinated and emanates from a site close to the junction of the superior mesenteric vein and splenogastric vein (Greenwood IA, Yeung SY, and Saleh SN, unpublished observations; see also Ref. 40). Recently, noncontractile cells with numerous filamentous projections analogous to the pacemaker cells seen in the gastrointestinal tract were observed in the rabbit PV (34). However, there is no evidence for these cells in murine PV (39), suggesting that the rhythmicity in this vessel is myogenic. Regardless of the underlying mechanism, the data in the present study add weight to the hypothesis that opening Cl channels by a rise in Ca2+ is a crucial element in gating the excitability of the PV. We show that NFA reduced the amplitude and frequency of spontaneous contractions at concentrations sufficient to inhibit IClCa but not to affect spasms produced by 60 mM KCl. Similar effects on spontaneous contractions were observed in rat PV (22). We also have shown the existence of NFA-sensitive afterdepolarizations in current-clamp mode using the perforated patch technique. Similar late depolarizations have been described in rat PV myocytes (31) and sheep lymphatic cells (41). The appearance of this late depolarized phase would maintain the cell in a more excitable state and would augment the degree of Ca2+ influx, consistent with an excitatory role of IClCa in smooth muscle cells. Our data support the earlier observation that PV from mice with NKCC1 knockouts exhibited less spontaneous activity (28).

Our data, taken together, allow the construction of a basic model whereby Ca2+ release from IP3-sensitive and IP3-insensitive Ca2+ stores occurs in response to Ca2+ influx through dihydropyridine-sensitive VDCC to produce a localized rise in [Ca2+] sufficient to open Ca2+-dependent Cl channels. The subsequent Cl ion efflux and membrane depolarization increase the open probability of VDCC. Because spontaneous contractions of murine PV were abolished by Ca2+-free bathing solution or dihydropyridines (present study and Ref. 39), influx of Ca2+ through VDCC appears to be the prime determinant of contraction. Consequently, changes in the activation of IClCa result in decreased contractility in the PV. The goals remain for future experiments to determine how Ca2+ released from intracellular stores contributes to the activation of spontaneous IClCa and to define the precise mechanism defining PV contractility.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. N. Saleh is sponsored by a Biotechnology and Biological Sciences Research Council studentship in collaboration with GlaxoSmithKline.


    ACKNOWLEDGMENTS
 
The help of Paul Andrews, Department of Basic Medical Sciences, Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, is gratefully acknowledged.


    FOOTNOTES
 

Address for reprint requests and other correspondence: I. A. Greenwood, Department of Basic Medical Sciences, Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, Jenner Wing, Cranmer Terrace, London SW17 0RE, United Kingdom (E-mail: i.greenwood{at}sghms.ac.uk)

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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 DISCUSSION
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