Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

In Deok Kong, Sang Don Koh, and Kenton M. Sanders

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to -50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca2+ with equimolar Mn2+ caused STOCs to "run down." Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs" remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca2+-activated K+ channels (SK channels). Application of ATP or 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, a P2 receptor blocker. Similarly, pretreatment of cells with U-73122 (1 µM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP3) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P2Y receptors results in localized Ca2+ release via PLC- and IP3-dependent mechanisms. Release of Ca2+ is coupled to STOCs, which are composed of currents mediated by large-conductance Ca2+-activated K+ channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

calcium sparks; small-conductance calcium-activated potassium channels; purinergic neurotransmission; P2Y receptors; inositol 1,4,5-trisphosphate receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MANY TYPES OF CELLS UNDERGO spontaneous, localized Ca2+ release (Ca2+ "sparks" or "puffs"), and local Ca2+ concentration can reach levels sufficient to regulate Ca2+-dependent conductances in the plasma membrane (13, 20). In the original communication describing Ca2+ sparks in smooth muscles, these events were reported to be coupled to spontaneous transient outward currents (STOCs; see Ref. 20), which are due to activation of large-conductance Ca2+-activated K+ (BK) channels (4). Recent experiments have shown that localized Ca2+ transients regulate the open probabilities of other Ca2+-dependent conductances, such as Ca2+-activated Cl- channels (31). In most studies of Ca2+ sparks in smooth muscles, localized Ca2+ release has been attributed to ryanodine receptors because treatment of cells with ryanodine blocked spontaneous and agonist-enhanced sparks (16, 20, 24, 31). Recently, however, release of Ca2+ from inositol 1,4,5-trisphosphate (IP3) receptor-operated stores has also been shown to be a source of Ca2+ spark activity in smooth muscles, and there may be a regenerative relationship between Ca2+ release from IP3 receptors and ryanodine receptors (3, 6).

Normally, enhanced production of IP3 and subsequent Ca2+ release is characteristic of responses to excitatory agonists in smooth muscles. However, localized Ca2+ transients mediated by IP3 receptors may provide a novel means by which Ca2+-dependent ionic conductances are activated by inhibitory agonists. In gastrointestinal (GI) smooth muscles, this mechanism might explain the inhibitory actions of ATP, which is thought to be an inhibitory neurotransmitter released from enteric motoneurons (11, 17). Stimulation of GI muscle cells with ATP or the P2Y receptor agonist 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) leads to activation of small-conductance Ca2+-activated K+ (SK) channels and hyperpolarization (18, 28). The inhibitory response to ATP in GI muscles appears to involve occupation of P2Y receptors (9, 29), activation of phospholipase C (PLC), and enhanced production of IP3 (2, 8, 21). It is possible that localized Ca2+ release could provide the link between P2Y receptors and activation of SK channels.

We previously showed that Ca2+ sparks in murine colonic muscles are elicited by IP3-dependent mechanisms (3), but STOCs, the standard electrophysiological assay of Ca2+ sparks, are due to activation of BK channels (see Ref. 20 and review in Ref. 7). BK channels do not mediate the inhibitory responses attributable to ATP in GI muscles, because inhibitors of BK do not block postjunctional inhibitory junction potentials (29). Purinergic inhibitory responses appear to be mainly due to activation of SK channels (18, 28). In the present study, we attempt to relate the P2Y receptor-IP3 pathway to activation of SK channels by investigating whether apamin-sensitive STOCs [not blocked by charybdotoxin (ChTX) or iberiotoxin] are present in GI muscles and regulated by purinergic agonists. We use myocytes isolated from the guinea pig taenia coli for these studies, because this is the classic preparation in which apamin-sensitive, enteric inhibitory responses attributed to ATP were first observed (1, 10, 27).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of smooth muscle cells. Guinea pigs of either sex were killed by CO2 asphyxiation. Isolated strips (1-2 cm long) of taenia coli were incubated in Ca2+-free Hanks' solution (see Solutions), containing 0.12% (wt/vol) collagenase (Worthington Biochemical, Freehold, NJ), 0.2% soybean trypsin inhibitor (type II-S, Sigma, St. Louis, MO), and 0.2% BSA (Sigma). After incubation at 37°C for 15 min with gentle stirring, tissues were washed four times with enzyme-free Hanks' solution. The tissue pieces were then triturated with a wide-bore fire-polished Pasteur pipette to create a cell suspension. The cells were stored at 4°C and used within 6 h.

Current and voltage measurements. Drops of the cell suspensions were placed on a glass coverslip forming the bottom of a 300-µl chamber mounted on an inverted microscope. Cells were allowed to adhere to the coverslip, and then the chamber was perfused at a rate of 3 ml/min. "Giga-seals" were made with fire-polished glass pipettes having tip resistances of 3-4 MOmega . The whole cell, perforated patch (amphotericin B) configuration of the patch-clamp technique was used to record ionic currents under voltage clamp. An Axopatch 200B amplifier with a CV-4 headstage (Axon Instruments, Foster City, CA) was used to measure ionic currents and membrane potentials. Current-clamp experiments (0 current) also were performed on cells with amphotericin-perforated patches. The changes of membrane potential were recorded on a chart recorder (Gould 2200S). A personal computer running pCLAMP software (version 6.0.4, Axon Instruments) was used to collect data.

Frequencies and amplitudes of STOCs were analyzed using the Mini analysis program (Synaptosoft Software, Leonia, NJ) for 100 s at given holding potentials. Amplitude histograms were constructed using a bin size of 1 pA. All recordings were performed at room temperature (22-25°C).

Solutions and reagents. CaCl2, KCl, KH2PO4, NaCl, NaHCO3, Na2HPO4, sucrose, and glucose were from Fisher Scientific (Fair Lawn, NJ). (±)Bay K 8644, xestospongin C, U-73122, and U-73343 were purchased from Calbiochem and dissolved in DMSO. After dilutions, the final concentration of DMSO was <0.01%. ATP and 2-MeS-ATP were purchased from Sigma. After the nucleotides were dissolved into HEPES-based buffer, the pH was corrected to 7.4. All other chemicals were also purchased from Sigma. Krebs solution contained (in mM) 125 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 Na2HPO4, and 11.5 glucose, with pH adjusted to 7.4 by bubbling with 95% O2 and 5% CO2. Ca2+-free Hanks' solution contained (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4, 0.44 KH2PO4, 10 glucose, 2.9 sucrose, and 11 HEPES, with pH adjusted to 7.4 with NaOH. The bath solution (CaPSS) contained (in mM) 135 NaCl, 5.0 KCl, 2.0 CaCl2, 1.2 MgCl2, 10.0 glucose, and 10 HEPES, with pH adjusted to 7.4 with Tris. The pipette solutions contained (in mM) 110 potassium gluconate, 30 KCl, 5 MgCl2, and 5 HEPES, with pH adjusted to 7.2 with Tris.

Statistical analyses. Data are expressed as means ± SE of n cells. All statistical analyses were performed using SigmaStat 2.0 software (Jandel, San Rafael, CA). We used paired t-tests and one-way repeated measures ANOVA to compare groups of data. In all statistical analyses, P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STOCs in isolated myocytes. Single myocytes from the guinea pig taenia coli were studied with the amphotericin-perforated patch technique. In preliminary studies, we noted that STOCs could be evoked when cells were held at depolarized potentials. STOCs were not resolved at potentials negative to -60 mV. Near -50 mV, small-amplitude STOCs (<10 pA) were recorded. When cells were held at more positive potentials, STOCs of highly variable amplitude and frequency were recorded (Fig. 1A). Amplitude histograms were constructed, and the peak currents of each distribution (1-pA bin size) were plotted as a function of membrane potential (Fig. 1, B and C). The amplitude distributions were bimodal: the mean amplitude of the large-amplitude STOCs was 19 ± 3 pA, and the mean amplitude of small-amplitude STOCs was 5 ± 1 pA, at a holding potential of -30 mV (n = 8). We refer to STOCs of <10 pA as "mini-STOCs" and STOCs of >10 pA as "large STOCs" at holding potentials of -40 mV or -30 mV.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of charybdotoxin (ChTX) on spontaneous transient outward currents (STOCs) in cells studied with amphotericin-permeabilized patch technique. A: control. Representative STOCs recorded at test potentials ranging from -50 to -20 mV. At holding potentials of -30 mV or -40 mV, mini-STOCs were defined as events <10 pA and large STOCs (denoted by *) as events >10 pA. Inset: expanded scale showing STOCs (arrows). At potentials more negative than -40 mV, it was not possible to distinguish between mini-STOCs and large STOCs, so all events are grouped as mini-STOCs. B: current-voltage (I-V) relationship summarizing STOCs from 8 cells. C: representative amplitude histogram of mini- and large STOCs recorded at a holding potential of -30 mV. Amplitude distribution shows a bimodal distribution. D: ChTX (200 nM; traces were recorded after 10-min incubation) abolished activity of large STOCs. Mini-STOCs were still apparent after ChTX (denoted by +). Inset: remaining mini-STOC in presence of ChTX. E: I-V relationship after treatment with ChTX (n = 8). F: representative amplitude histogram (holding potential -30 mV) shows inhibition of large STOCs and reduction in mini-STOCs after ChTX treatment.

STOCs have been reported to result from openings of BK channels, and these events are initiated by focal release of Ca2+ (Ca2+ sparks) from intracellular stores (4, 20). STOCs of varying amplitudes could be due to multiple Ca2+ spark sites where different numbers of BK channels are affected or due to multiple populations of Ca2+-dependent conductances. We tested the latter hypothesis with channel antagonists to selectively block specific conductances. Addition of ChTX (200 nM; n = 5), iberiotoxin (200 nM; n = 3), or TEA (1 mM; n = 2), potent inhibitors of BK channels, completely blocked large-amplitude STOCs (Fig. 1, D-F). ChTX also attenuated mini-STOCs from 338 ± 59 to 60 ± 8 events/100 s (82% inhibition) at a holding potential of -30 mV (Figs. 1 and 2). These data suggest that mini-STOCs may be partially due to BK channels, but a ChTX-resistant component also contributed to mini-STOCs. ChTX-resistant mini-STOCs were completely abolished within 10 min when extracellular Ca2+ was replaced with equimolar Mn2+ (Fig. 2C), and STOC activity recovered when extracellular Ca2+ was restored (data not shown).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of extracellular Ca2+ on STOCs. Same cell was held at -30 mV. A: STOCs recorded under control conditions (i.e., 2 mM external Ca2+ concentration). B: mini-STOCs remaining in presence of 200 nM ChTX for 10 min. C: inhibition of all STOCs after replacement of external Ca2+ in Ca2+-containing physiological saline solution (CaPSS) with Mn2+ (MnPSS). D: summary of results from 5 cells expressing number of events during 100-s recordings. Data are grouped in 10-pA bins. * P < 0.05.

Bay K 8644 (2 µM) markedly increased both types of STOCs (Fig. 3, A and B), but nicardipine (1 µM) did not affect the occurrence of STOCs (data not shown). Exposing cells to caffeine (1 mM) caused large "burstlike" episodes of STOCs. The enhanced STOC activity was followed by a quiescent period (holding potential -40 mV; Fig. 3, C and D). Ryanodine (10 µM) did not significantly affect the occurrence of either type of STOC (holding potential -30 mV; n = 4; Fig. 4, A-C). In contrast, xestospongin C (1 µM), a membrane-permeant blocker of IP3 receptors (14), significantly inhibited both large STOCs and mini-STOCs (n = 4; Fig. 4, D-F). These data suggest that STOCs are not caused by, but can be enhanced by, Ca2+ entry via L-type Ca2+ channels. The trigger for STOCs in these cells appears to be release of Ca2+ from an IP3 receptor-operated store.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of Bay K 8644 and caffeine on STOCs. Cells were held at holding potential of -40 mV. A: control. STOCs recorded in normal CaPSS. B: after application of Bay K 8644 (2 µM). C: control. STOCs recorded from a different cell in normal CaPSS. D: after application of caffeine (1 mM). Parallel lines denote 2 min or recording omitted between 5th and 6th traces.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of ryanodine and xestospongin C on STOCs. Cells were held at -30 mV. A: control. STOCs recorded in normal CaPSS. B: after addition of ryanodine (10 µM) for 20 min. C: summary of results showing number of events during 100-s recordings from 4 cells. D: control. STOCs in a different cell in CaPSS. E: after addition of xestospongin C (1 µM) for 20 min. F: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05 vs. control.

Apamin (500 nM), an antagonist of SK channels (5, 12, 18), attenuated mini-STOCs (Fig. 5). Mini-STOCs recorded during 100 s were reduced from 373 ± 32 to 306 ± 23 events (17.4% inhibition, n = 5, P < 0.05). Large STOCs were also slightly decreased by apamin treatment; however, this effect did not reach a level of statistical significance (Fig. 5, B and C). It is possible that large-amplitude STOCs contain a component of current contributed by apamin-sensitive channels. Inhibition of these channels may cause reduction in the amplitudes of large STOCs and cause these events to be tabulated as mini-STOCs after apamin. These experiments were conducted at a holding potential of -30 mV to exclude the potential involvement of Ca2+-activated Cl- currents (expected reversal potential -40 to -30 mV). The data suggest that STOCs in colonic myocytes result from transient openings of BK and apamin-sensitive SK channels.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of apamin on STOCs. Cell was held at -30 mV. A: control. STOCs in CaPSS. B: after addition of apamin (500 nM) for 10 min. C: summary of results showing number of events during 100-s recordings from 5 cells. * P < 0.05 vs. control.

Activation of STOCs by ATP and 2-MeS-ATP. Purinergic stimulation by ATP and 2-MeS-ATP activates SK channels in visceral smooth muscle (18, 28). We tested the effects of these agonists at a holding potential of -40 mV. ATP (1 mM; n = 4) and 2-MeS-ATP (100 µM; n = 5) significantly increased the activity of mini-STOCs (control vs. ATP, from 204 ± 25 to 273 ± 25 events/100 s, a 35% increase; control vs. 2-MeS-ATP, from 174 ± 27 to 257 ± 39 events/100 s, a 50% increase; Fig. 6). Large STOCs also tended to be increased by ATP (from 10 ± 6 to 23 ± 12 events/100 s) and 2-MeS-ATP (from 14 ± 8 to 28 ± 14 events/100 s); however, these responses did not reach levels of statistical significance. We also characterized purinergic activation of STOCs in the presence of ChTX to block BK channels. As above, ChTX (200 nM) significantly attenuated large STOCs and mini-STOCs in these cells (Fig. 7, A and B). Addition of 2-MeS-ATP in the continued presence of ChTX increased the frequency (ChTX vs. ChTX + 2-MeS-ATP, 46 ± 12 vs. 122 ± 15, 222% increase, n = 4) of mini-STOCs (Fig. 7, C and D). Both components of STOCs were greatly attenuated by joint application of apamin and ChTX (i.e., mini-STOCs were reduced from 313 ± 30 to 16 ± 4 events/100 s by apamin and ChTX, a 95% inhibition; Fig. 8, A and B). Large STOCs were completely abolished by apamin and ChTX. In the presence of these drugs, 2-MeS-ATP did not increase the activity of mini-STOCs or large STOCs (Fig. 8, C and D). These data suggest that at least a portion of the response to purinergic stimulation is due to STOCs mediated through activation of apamin-sensitive channels.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of ATP and 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) on STOCs. Cells were held at -40 mV. A: control STOCs. B: after addition of ATP (1 mM). C: summary of results showing number of events during 100-s recordings from 4 cells. D: control. STOCs from a different cell. E: after addition of 2-MeS-ATP (100 µM). F: summary of results showing number of events during 100-s recordings from 5 cells. * P < 0.05 vs. control.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of 2-MeS-ATP on STOCs in presence of ChTX. Cell was held at -30 mV. A: control. STOCs in normal CaPSS. B: after addition of ChTX (200 nM) for 10 min. C: 2-MeS-ATP (100 µM) increased number of mini-STOCs in presence of ChTX. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 8.   Effects of 2-MeS-ATP on STOCs in presence of ChTX and apamin. Cell was held at -30 mV. A: control. STOCs in normal CaPSS. B: after addition of ChTX (200 nM) and apamin (500 nM) for 10 min. C: in presence of ChTX and apamin, 2-MeS-ATP (100 µM) failed to increase STOCs. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

Receptors and second messengers mediating the effect of 2-MeS-ATP on STOCs. We tested whether the enhancements in STOCs by ATP and 2-MeS-ATP were due to activation of purinergic receptors. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 5 µM), an antagonist of P2 receptors (19), decreased large STOCs and mini-STOCs at a holding potential of -30 mV (Fig. 9, A and B). 2-MeS-ATP failed to increase the activity of STOCs in the presence of PPADS (Fig. 9, C and D; n = 4).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 9.   Effects of 2-MeS-ATP on STOCs in presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS). Cell was held at -30 mV. A: control. STOCs in normal CaPSS. B: after addition of PPADS (5 µM, 10-min incubation). C: in presence of PPADS, 2-MeS-ATP (100 µM) failed to increase STOCs. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

Cells were pretreated with U-73122 (1 µM), an inhibitor of PLC (see Ref. 25), for 10 min to determine whether the activation of PLC mediates the increase in STOCs caused by 2-MeS-ATP. U-73122 decreased the activity of mini-STOCs (from 304 ± 27 to 224 ± 24 events/100 s, 26% inhibition, n = 4; Fig. 10). 2-MeS-ATP (100 µM) failed to increase the activity of STOCs in the presence of this compound (Fig. 10, C and D; n = 4). U-73343 (1 µM), an inactive form of U-73122, had no effect on STOCs, and 2-MeS-ATP increased mini-STOCs in the presence of U-73343 (holding potential -30 mV; Fig. 11; n = 4). 2-MeS-ATP failed to enhance STOC frequency in cells pretreated with xestospongin C (n = 2).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 10.   Effects of 2-MeS-ATP on STOCs in presence of U-73122. Cell was held at -30 mV. A: control. STOCs in normal CaPSS. B: after addition of U-73122 (1 µM, 10-min incubation). C: 2-MeS-ATP (100 µM) failed to increase STOCs in presence of U-73122. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 11.   Effects of 2-MeS-ATP on STOCs in presence of U-73343. Cell was held at -30 mV. A: control. STOCs in normal CaPSS. B: after addition of U-73343 (1 µM, 10-min incubation). C: 2-MeS-ATP (100 µM) increased mini-STOCs in presence of U-73343. D: summary of results showing number of events during 100-s recordings from 4 cells. * P < 0.05.

Hyperpolarization induced by purinergic activation. In the current-clamp mode (0 current), we investigated the effects of 2-MeS-ATP on membrane potential. At rest, membrane potential averaged -44.2 ± 3.9 mV (n = 7). In three of seven cells, small fluctuations in resting membrane potential were observed during resting conditions. Application of 2-MeS-ATP (100 µM) to these cells induced transient hyperpolarizations (10 ± 3 mV in amplitude, n = 3; Fig. 12A). There was variability in the durations of the transient hyperpolarizations, which may have reflected differences in the degree of summation of STOCs. In four cells, spontaneous spikelike hyperpolarizations were observed during resting conditions (e.g., frequency averaged 353 ± 29 per 100 s). Addition of ChTX (200 nM) under current-clamp conditions induced a 3 ± 1 mV depolarization (P < 0.05) and decreased the amplitude (to 5 ± 2 mV, P < 0.05) and frequency (to 220 ± 23 per 100 s, P < 0.05; see Fig. 12B) of the transient hyperpolarizations. In the presence of ChTX, 2-MeS-ATP restored the amplitude of the spontaneous transient hyperpolarizations (to 9 ± 2 mV, P < 0.05 compared with amplitude in the presence of ChTX) and increased the frequency (to 580 ± 46, P < 0.05; see Fig. 12B). These events were consistent with the changes in STOC frequency induced by ChTX and 2-MeS-ATP under voltage-clamp conditions. The data suggest that the hyperpolarizations caused by 2-MeS-ATP are mediated, in part, by ChTX-insensitive STOCs.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 12.   Effects of 2-MeS-ATP on resting membrane potentials of current-clamped cells (0 current). A: continuous trace from a representative cell showing transient hyperpolarization after application of 2-MeS-ATP (100 µM; exposure denoted by bar). B: discontinuous recording (10 min were removed between 1st and 2nd traces, as denoted by parallel lines at end of 1st trace) demonstrating effects of 2-MeS-ATP after prior addition of ChTX (200 nM). ChTX (black bar over 1st and 2nd traces) reduced amplitude and frequency of transient hyperpolarizations that occurred spontaneously in this cell and produced a small depolarization in membrane potential. In presence of ChTX, 2-MeS-ATP (shaded bar in 2nd trace) increased frequency of transient hyperpolarizations and restored membrane potential. Dotted line denotes average resting potential during control phase of recording.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STOCs have been used as an assay of localized Ca2+ release (4, 20), and quantitative analysis has shown a high degree of correlation between Ca2+ spark amplitude and STOC amplitude (22). In some smooth muscle cells, however, Ca2+ sparks are coupled to more than a single Ca2+-dependent conductance (31), and the present study indicates that this is also the case in guinea pig taenia coli smooth muscle cells. Records of STOCs from taenia coli myocytes showed typical large-amplitude STOCs and frequent low-amplitude (<10 pA) STOC-like events. All STOCs were Ca2+ dependent; they were enhanced by Bay K 8644 and disappeared when cells were incubated for extended periods in low external Ca2+ or after intracellular stores were depleted by addition of caffeine. The data indicated that Ca2+ release from IP3-sensitive stores may be the trigger for STOCs in taenia coli myocytes. When BK channels were blocked by ChTX or iberiotoxin (by addition of >15 times the dissociation constant for smooth muscle BK channels; see Refs. 15, 23), STOC-like activity persisted, but the remaining events were of much smaller amplitude than the events attributable to BK. Mini-STOCs were partially blocked by apamin, suggesting that at least a portion of the current was due to SK channels. These data suggest that at least two Ca2+-dependent conductances are activated by localized Ca2+ release in myocytes from the taenia coli.

The mini-STOCs were relatively difficult to resolve in relation to STOCs caused by BK channels. This is likely due to the fact that the conductance of SK channels is comparatively low (i.e., 5.3 pS, see Ref. 18). Thus >40 SK channels would need to open at the same time to produce the conductance equivalent to 1 BK channel. Because STOCs are typically attributed to the nearly simultaneous increase in open probability of many BK channels, a very large number of SK channels would need to be clustered near sites of Ca2+ sparks to produce STOCs of an amplitude equivalent to BK STOCs. Thus it is logical that STOCs due to SK channels are of much smaller amplitude and typically obscured by the much larger amplitude BK STOCs. Typically, openings of SK channels might enhance the amplitude of BK STOCs or produce what would appear to be increased baseline noise in current records. The need to use depolarized potentials to resolve the small currents associated with mini-STOCs increases contamination from BK channels. Therefore, resolution of STOCs due to SK channels required blocking of BK channels.

It was not possible to separate STOCs due to SK and BK channels purely on the basis of amplitude. A significant portion (~80%) of the mini-STOCs were inhibited by ChTX, suggesting that smaller clusters of BK channels or clusters at some distance from the primary spark sites may contribute to the low-amplitude population of STOCs. Apamin inhibited the small-amplitude STOCs by ~20%. Therefore, the mini-STOCs appear to represent outward currents due to openings of both BK and apamin-sensitive SK channels.

The mechanisms by which ATP increases cell membrane conductance and causes hyperpolarization of GI smooth muscles have been unclear. Previous studies showed that ATP, UTP, and the P2Y agonist 2-MeS-ATP activated an outward current under whole cell recording conditions, and this was attributed to an increase in the open probability of SK channels (18, 28). In the present study, we show that 2-MeS-ATP increases the frequency of STOCs, and the data suggest that this occurs via occupation of P2Y receptors (PPADS), activation of PLC (U-73122), and increased IP3 production (xestospongin C). The increase in STOCs in response to 2-MeS-ATP suggests that IP3-dependent amplification of Ca2+ release (either an increase in the number of sparking sites or in the frequency of sparking from established sites) is the main mechanism responsible for P2Y receptor-mediated stimulation of outward current and hyperpolarization. Experiments performed in current clamp clearly demonstrate the link between purinergic stimulation and hyperpolarization. In isolated cells, hyperpolarization transients were quantal in nature and most likely the result of the STOCs recorded under voltage clamp. In intact tissues, the quantal hyperpolarization transients occurring in many coupled cells would tend to summate temporally and spatially, yielding continuous, analog hyperpolarization responses.

We found that PPADS (P2 receptor blocker) and U-73122 (PLC blocker) also reduce the frequency of spontaneous STOCs. These data could be interpreted as nonspecific effects of these compounds on SK channels. Previous studies argue against the idea that PPADS blocks SK channels. We found that PPADS decreased the open probability of SK channels in murine colon, but these channels could be activated by releasing internal stores of Ca2+ with caffeine (18). PPADS has been shown to be a partial antagonist of IP3 receptors (26), and this may explain why this compound reduced spontaneous STOC activity. The nonspecific effects of U-73122 were controlled for by also testing U-73343, a nonactive, structurally similar analog of U-73122. The inactive analog had no effect on STOC occurrence. It is likely that U-73122 reduced basal production of IP3 in the cells, and this might explain the reduction in STOC frequency in response to this compound.

In studies of mouse colonic myocytes, we showed that spontaneous Ca2+ sparks are caused by release of Ca2+ from IP3 receptor-operated stores (3). Purinergic stimulation increased Ca2+ sparks and tended to organize local release events into Ca2+ waves. Ca2+ sparks and Ca2+ waves never elevated global Ca2+ to the threshold for contraction. A similar enhancement in Ca2+ spark frequency in response to 2-MeS-ATP is also likely in taenia coli, because we noted an increase in STOCs when cells were exposed to this compound.

Ca2+ release is associated with activation of SK and BK channels in myocytes from the taenia coli. This raises the question of why tissue responses to purinergic stimuli or enteric inhibitory neural inputs are reduced by apamin but unaffected by inhibition of BK channels (30). As was recently pointed out by ZhuGe and co-workers (31), the ionic conductances activated by Ca2+ sparks are strongly affected by the voltage range in which cells are held or are operating during physiological conditions. These authors found that BK STOCs were favored at more depolarized potentials, but Ca2+-activated Cl- currents (spontaneous transient inward currents) were more frequently observed at more negative potentials. These results are partly explained by the driving forces responsible for outward K+ currents and inward Cl- currents. However, BK channels are both voltage and Ca2+ dependent, and the open probability of these channels is very low at the relatively negative resting membrane potentials of GI muscles (i.e., negative to -50 mV). SK channels are not voltage dependent, and open probability is increased by low concentrations of Ca2+ (18). Therefore, the weighting of purinergic enteric inhibitory responses toward SK channels may be partially due to the voltage range of resting membrane potentials of GI muscles. Stimulating cells with 2-MeS-ATP yielded increases mainly in mini-STOCs, suggesting that Ca2+ release via IP3 receptor-operated stores occurs in close proximity to clusters of SK channels. Therefore, it is possible that part of the discrimination between activation of SK and BK channels is due to tight coupling between receptors, second messenger pathways, IP3 receptor-operated stores, and SK channels. It would be extremely interesting to know whether the clustering of cellular effector proteins are postjunctional specializations associated with sites of neural innervation in intact muscles.

In summary, the present study suggests a mechanism by which purinergic neurotransmission causes hyperpolarization and reduces the excitability of GI smooth muscles. The data are consistent with the following. Occupation of P2Y receptors activates PLC and increases production of IP3. Rather than generally increasing global cytoplasmic Ca2+, IP3 generated via this mechanism enhances localized Ca2+ transients. This leads to enhanced production of STOCs, which are mainly caused by activation of SK channels. STOCs cause hyperpolarization transients that summate and cause the hyperpolarization responses in intact muscles. Hyperpolarization of GI muscles lowers excitability and decreases the net open probability of L-type Ca2+ channels, leading to a net inhibitory effect.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant PO1-DK-41315.


    FOOTNOTES

Address for reprints and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557 (E-mail: kent{at}physio.unr.edu).

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. §1734 solely to indicate this fact.

Received 4 June 1999; accepted in final form 24 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Banks, B. E., C. Brown, G. M. Burgess, G. Burnstock, M. Claret, T. M. Cock, and D. H. Jenkinson. Apamin blocks certain neurotransmitter-induced increase in potassium permeability. Nature 282: 415-417, 1979[ISI][Medline].

2.   Barnard, E. A., T. E. Webb, J. Simon, and S. P. Kunapuli. The diverse series of recombinant P2Y purinoceptors. Ciba Found. Symp. 198: 166-180, 1996[ISI][Medline].

3.  Bayginov, O., B. Hagan, and K. M. Sanders. The inhibitory effects of ATP are mediated by localized calcium transients in murine colonic muscle. Gastroenterology. In press.

4.   Benham, C. D., and T. B. Bolton. Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J. Physiol. (Lond.) 381: 385-406, 1986[Abstract].

5.   Blatz, A. L., and K. L. Magleby. Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. Nature 323: 718-720, 1986[ISI][Medline].

6.   Boittin, F. X., F. Coussin, N. Macrez, C. Mironneau, and J. Mironneau. Inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channel-dependent Ca2+ signalling in rat portal vein myocytes. Cell Calcium 23: 303-311, 1998[ISI][Medline].

7.   Bolton, T. B., S. A. Prestwich, A. V. Zholos, and D. V. Gordienko. Excitation-contraction coupling in gastrointestinal and other smooth muscles. Annu. Rev. Physiol. 61: 85-115, 1999[ISI][Medline].

8.   Boyer, J. L., C. P. Downes, and T. K. Harden. Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides. J. Biol. Chem. 264: 884-890, 1989[Abstract/Free Full Text].

9.   Bultmann, R., O. Dudeck, and K. Starke. Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli. Naunyn Schmiedebergs Arch. Pharmacol. 353: 445-451, 1996[ISI][Medline].

10.   Burnstock, G., G. Campbell, M. Bennett, and M. E. Holaman. Inhibition of smooth muscle of the taenia coli. Nature 200: 581-582, 1963[ISI].

11.   Bywater, R. A., and G. S. Taylor. Non-cholinergic excitatory and inhibitory junction potentials in the circular smooth muscle of the guinea-pig ileum. J. Physiol. (Lond.) 374: 153-164, 1986[Abstract].

12.   Capiod, T., and D. C. Ogden. The properties of calcium-activated potassium ion channels in guinea-pig isolated hepatocytes. J. Physiol. (Lond.) 409: 285-295, 1989[Abstract].

13.   Cheng, H., W. J. Lederer, and M. B. Cannell. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740-744, 1993[ISI][Medline].

14.   Gafni, J., J. A. Munsch, T. H. Lam, M. C. Catlin, L. G. Costa, T. F. Molinski, and I. N. Pessah. Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 19: 723-733, 1997[ISI][Medline].

15.   Giangiacomo, K. M., M. Garcia-Calvo, K. Hans-Gunther, T. J. Mullmann, M. L. Garcia, and O. McManus. Functional reconstitution of the large-conductance, calcium-activated potassium channel purified from bovine aortic smooth muscle. Biochemistry 34: 15849-15862, 1995[ISI][Medline].

16.   Gordienko, D. V., T. B. Bolton, and M. B. Cannell. Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells. J. Physiol. (Lond.) 507: 707-720, 1998[Abstract/Free Full Text].

17.   Hoyle, C. V. H., and G. Burnstock. Neuromuscular transmission in the gastrointestinal tract. In: Handbook of Physiology. The Gastrointestinal System. Neural and Endocrine Biology. Bethesda, MD: Am. Physiol. Soc, 1989, sect. 6, vol I, chapt. 13, p. 435-464.

18.   Koh, S. D., G. M. Dick, and K. M. Sanders. Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle. Am. J. Physiol. Cell Physiol. 273: C2010-C2021, 1997[Abstract/Free Full Text].

19.   Lambrecht, G., T. Friebe, U. Grimm, U. Windscheif, E. Bungardt, C. Hildebrandt, H. G. Baumert, G. Spatz-Kumbel, and E. Mutschler. PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses. Eur. J. Pharmacol. 217: 217-219, 1992[ISI][Medline].

20.   Nelson, M. T., H. Cheng, M. Rubart, L. F. Santana, A. D. Bonev, H. J. Knot, and W. J. Lederer. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

21.   Nicholas, R. A., E. R. Lazarowski, W. C. Watt, Q. Li, J. Boyer, and T. K. Harden. Pharmacological and second messenger signalling selectivities of cloned P2Y receptors. J. Auton. Pharmacol. 16: 319-323, 1996[ISI][Medline].

22.   Perez, G. J., A. D. Bonev, J. B. Patlak, and M. T. Nelson. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J. Gen. Physiol. 113: 229-238, 1999[Abstract/Free Full Text].

23.   Perez, G. J., L. Toro, S. D. Erulkar, and E. Stefani. Characterization of large-conductance, calcium-activated potassium channels from human myometrium. Am. J. Obstet. Gynecol. 168: 652-660, 1993[ISI][Medline].

24.   Sieck, G. C., M. S. Kannan, and Y. S. Prakash. Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells. Can. J. Physiol. Pharmacol. 75: 878-888, 1997[ISI][Medline].

25.   Smith, R. J., L. M. Sam, J. M. Justen, G. L. Bundy, G. A. Bala, and J. E. Bleasdale. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J. Pharmacol. Exp. Ther. 253: 688-697, 1990[Abstract].

26.   Vigne, P., P. Pacaud, V. Urbach, E. Feolde, J. P. Breittmayer, and C. Frelin. The effect of PPADS as an antagonist of inositol (1,4,5)triphosphate induced intracellular calcium mobilization. Br. J. Pharmacol. 119: 360-364, 1996[Abstract].

27.   Vladimirova, I. A., and M. F. Shuba. [Effect of strychnine, hydrastine and apamin on synaptic transmission in smooth muscle cells]. Neirofiziologiia 10: 295-299, 1978[Medline].

28.   Vogalis, F., and R. K. Goyal. Activation of small conductance Ca2+-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum. J. Physiol. (Lond.) 502: 497-508, 1997[Abstract].

29.   Zagorodnyuk, V., and C. A. Maggi. Pharmacological evidence for the existence of multiple P2 receptors in the circular muscle of guinea-pig colon. Br. J. Pharmacol. 123: 122-128, 1998[Abstract].

30.   Zagorodnyuk, V., P. Santicioli, C. A. Maggi, and A. Giachetti. The possible role of ATP and PACAP as mediators of apamin sensitive NANC inhibitory junction potentials in circular muscle of guinea-pig colon. Br. J. Pharmacol. 119: 779-786, 1996[Abstract].

31.   ZhuGe, R., S. M. Sims, R. A. Tuft, K. E. Fogarty, and J. V. Walsh, Jr. Ca2+ sparks activate K+ and Cl- channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J. Physiol. (Lond.) 513: 711-718, 1998[Abstract/Free Full Text].


Am J Physiol Cell Physiol 278(2):C352-C362
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society