Calcium Release and Calcium-Activated Chloride Channels in Airway Smooth Muscle Cells

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1 Calcium Release and Calcium-Activated Chloride Channels in Airway Smooth Muscle Cells MICHAEL I. KOTLIKOFF and YONG-XIAO WANG Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Rapid progress has been made in the determination of specific ion channels expressed in airway smooth muscle cells and their role in excitation contraction coupling. The combination of molecular biology and molecular physiology has provided insight into the properties of voltage-dependent cation (calcium and potassium) channels and their regulation by excitatory and inhibitory signaling processes. In this brief review, we will focus on calcium release and calcium-activated chloride channels. The former channels mediate receptor-activated calcium release, and the latter channels are opened following this release event. Moreover, the discovery of spontaneous calcium release events, or calcium sparks, in smooth muscle, suggests an unanticipated level of regulation. Intracellular calcium release can drive electrical activity by the activation of calcium-dependent sarcolemmal ion channels, including calcium-activated chloride channels. These channels activate briefly but undergo a rapid phosphorylation by calcium/calmodulin-dependent protein kinase, which uncouples channel activity from cytosolic calcium. The coupling between intracellular calcium release and depolarizing chloride currents represents a potentially important signaling system in airway smooth muscle. Kotlikoff MI, Wang Y-X. Calcium release and calcium-activated chloride channels in airway smooth muscle cells. AM J RESPIR CRIT CARE MED 1998;158:S109 S114. Ion channels are key excitability proteins that control electrical signaling across cellular membranes. Channels provide a gated, aqueous pore that controls the permeation of specific ions across these membranes, thus altering the transmembrane voltage and, in the case of calcium ions, the concentration of the most important intracellular cellular signaling molecule. By understanding the mechanism by which these signals are generated, we begin to dissect the processes by which airway myocytes, the individual effectors of bronchospasm, are activated by upstream pathways. This process is particularly relevant in the asthmatic airway, where airway inflammation results in the release of a diverse array of bronchoactive components, the actions of which are poorly understood. Sarcolemmal and sarcoplasmic reticulum (SR) ion channels subserve several critical functions in excitation/contraction coupling. The latter channels include the IP 3 receptor and the ryanodine receptor, calcium permeant ion channels with similar structures that mediate the release of calcium into the cytosol during excitation. The release of calcium from the SR activates calcium-sensitive membrane sarcolemmal ion channels, such as calcium-activated chloride and calcium-activated potassium channels. In this paper we describe specific features of calcium release and the activation of calcium-activated chloride channels in airway smooth muscle cells (1 4). Correspondence and requests for reprints should be addressed to Michael I. Kotlikoff, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA Am J Respir Crit Care Med Vol 158. pp S109 S114, 1998 Internet address: CALCIUM RELEASE IN AIRWAY SMOOTH MUSCLE CELLS Calcium flux into the cytosol from the sarcoplasmic reticulum is mediated by two important calcium permeant channels in myocytes: the inositol trisphospate receptor (InsP 3 R) and the ryanodine receptor (RYR), which mediate calcium-induced calcium release (CICR). These ion channels share substantial sequence homology, protein topology, and ionic permeation properties (5 8). Both channels are assembled as tetramers and form nonselective cation channels; the high calcium gradient across the SR membrane results in a large calcium flux from the SR to the cytosol upon channel opening. A well-established action of neurotransmitter-activated excitation contraction coupling in smooth muscle is the phospholipase C mediated rise in InsP 3 concentration and opening of InsP 3 R. The InsP 3 receptor isolated from smooth muscle is a 224-kilodalton (kd) protein that binds InsP 3 with a dissociation constant (K) of approximately 2.4 nm (6). Based on the cloning of complementary DNA (cdna) isolated from various tissues, at least three isoforms of InsP 3 R exist (9 11), and all three receptor subtypes are expressed in most tissues, including smooth muscle (12, 13). Similarly, three isoforms of RYR have been identified, and RYR2 and RYR3 are the isoforms that likely underlie calcium release in smooth muscle (14). Calcium release through InsP 3 R is substantially slower than CICR because the calcium flux associated with a single opening of the InsP 3 R is more than 20-fold less than for an RYR opening (15), and CICR requires no enzymatic second messenger synthesis. The substantially larger calcium flux through RYR receptors may also be important for coupling to calcium-sensitive sarcolemmal ion channels (16), as discussed below. Measurements of intracellular calcium in single, voltageclamped smooth muscle cells has provided a powerful method

2 S110 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL to examine the mechanisms and sites of intracellular calcium release in smooth muscle. We have investigated the release of calcium following muscarinic receptor stimulation and the attendant activation of calcium-activated chloride currents (I Cl(Ca) ) in voltage-clamped equine tracheal myocytes (1). Single isolated airway myocytes are loaded with Fura-2 and voltage-clamped using the perforated patch clamp technique; the pipette solution often contains Cs intracellular solution to block potassium currents. Under these conditions, stimulation of cells with muscarinic acetylcholine (mach) (50 M) or release of intracellular calcium with caffeine (8 mm) produces markedly different [Ca 2 ] i and current responses. As shown in Figure 1, a transient Ca 2 -activated Cl current (I Cl(Ca) ) is activated by both stimuli, whereas muscarinic stimulation activates an additional nonselective cation current that is not activated by caffeine (2). The identity of the currents has been confirmed using voltage ramps to determine the instantaneous current reversal potential; also, in cells dialyzed with 10 mm EGTA/ 1 mm Ca 2, mach or caffeine application fails to evoke a rise in calcium or a transient current. These data indicate that caffeine and mach activate I Cl(Ca) consistent with previous descriptions of the muscarinic activation of I Cl(Ca) in anococcygeus (17), intestinal (18), and tracheal smooth muscle (19). In order to examine the mechanism of calcium release in airway smooth muscle, single myocytes were dialyzed with heparin or ruthenium red to selectively block IP 3 receptors or ryanodine receptors, respectively (20). Fura-2 (75 M) was included in the patch pipette solution for the measurement of [Ca 2 ] I. As shown in Figure 2, following intracellular dialysis of heparin (10 mg ml 1 ) for 5 min, application of mach (50 M) failed to induce any [Ca 2 ] I increase or membrane current. Subsequent application of caffeine (8 mm) to the same cell, however, induced a typical increase in [Ca 2 ] I and I Cl(Ca). In a series of similar experiments the peak [Ca 2 ] I increase and I Cl(Ca) were nm (resting nm) and 1, picoamperes (pa) in myocytes predialyzed with heparin (n 6), and nm (resting nm) and pa in control cells (n 7). Cells were also dialyzed with IP 3 (100 M), followed by exposure to mach (50 M). Within 1 2 min after establishment of the whole-cell configuration, I Cl(Ca) was activated when the patch pipette solution contained IP 3, and the current was similar in size and time course to the mach response (1). Moreover, application of mach (50 M) failed to induce I Cl(Ca) after IP 3 dialysis. Similar results were obtained in six other cells tested. Thus, heparin or dialysis with IP 3 abolished calcium release associated with muscarinic stimulation, although heparin had no effect on the ability of caffeine to effect calcium release. To further examine whether ryanodine receptor-mediated [Ca 2 ] I release is associated with muscarinic activation of I Cl(Ca), experiments were carried out in cells dialyzed with ruthenium red (50 M). Figure 3A shows an example of these experiments. To test for adequacy of ryanodine receptor block, caffeine (8 mm) was applied after dialysis of ruthenium red for 5 min; caffeine did not evoke an increase in [Ca 2 ] i or I Cl(Ca), indicating functional block of ryanodine receptors. Subsequent application of mach (50 M), however, induced a typical [Ca 2 ] i increase and I Cl(Ca), which were not significantly different from responses obtained in cells dialyzed without ruthenium red. The rate of rise of [Ca 2 ] i was also quite similar between the groups. If caffeine was used to release calcium through ryanodine receptors, however, muscarinic stimulation produced no Figure 1. Muscarinic stimulation and calcium release evoke distinct currents and intracellular calcium responses in single tracheal myocytes. Repeated exposure of a cell to mach activates a transient calcium-activated chloride current (I Cl(Ca) ) and a sustained, noisy cation current (top). The [Ca 2 ] i response is markedly biphasic and shows a sustained increase in [Ca 2 ] i. The 150-ms voltage ramps from 60 to 50 mv were used to identify the current reversal potentials. Release of intracellular calcium with caffeine results in a transient increase in [Ca 2 ] i that promptly returns to baseline, and a calcium-activated chloride current, but no cation current (bottom). Figure 2. IP 3 receptor mediated calcium release underlies muscarinic stimulation of I Cl(Ca). Block of InsP 3 receptors with heparin (dialyzed for 5 min) prevents calcium release and activation of I Cl(Ca) by methacholine. Application of caffeine releases calcium through activation of ryanodine receptors, and activates the current.

3 Kotlikoff and Wang: Calcium Release in Airway Smooth Muscle Cells subsequent calcium release or I Cl(Ca), whereas after washout of caffeine (5 min) mach induced a typical response (Figure 3B). Moreover, the release of intracellular calcium and activation of I Cl(Ca) by mach prevented a subsequent calcium release and calcium-activated chloride current when caffeine was applied immediately following the termination of the mach application (1). Taken together, these results suggest that IP 3 receptors and ryanodine receptors are functionally coupled to the same intracellular calcium stores in airway smooth muscle, and that IP 3 -mediated calcium release is sufficient to explain the muscarinic increase in [Ca 2 ] I and activation of I Cl(Ca). The results also suggest that global calcium-induced calcium release through ryanodine receptors does not appear to play a prominent role in muscarinic stimulation of airway myocytes. However, although muscarinic release of intracellular calcium, at least at high agonist concentrations, does not appear to rely on CICR for full calcium release, this result should not be taken S111 to suggest that CICR does not exist in airway smooth muscle. Using laser scanning confocal microscopy, we have directly observed calcium sparks analagous to those observed in cardiac (21, 22), skeletal (23), and vascular smooth muscle (16). These quantal calcium release events result from the activation of ryanodine receptors even at resting [Ca 2 ] i, and are coupled to the activation of calcium-activated ion channels. Thus, it should be emphasized that while the probability of opening of RYR is low, it is not zero, and the stochastic gating of these channels and the attendant calcium release can have dramatic effects on cell electrical activity. An example of this activity is the spontaneous transient inward currents (STICs) that are observed in airway smooth muscle cells. These depolarizing currents dominate electrical activity in airway smooth muscle cells at potentials negative to approximately 40 mv, as shown in Figure 4. They are almost certainly activated by local calcium sparks, as has been demonstrated for spontaneous outward currents (16). RELATIONSHIP BETWEEN [Ca 2 ] i AND I Cl(Ca) To determine the dependence of I Cl(Ca) on [Ca 2 ] i, the instantaneous relationship between I Cl(Ca) and [Ca 2 ] i was examined using high time resolution calcium measurements. As shown in Figure 5, the time course of the increase in [Ca 2 ] i was quite similar to that of I Cl(Ca), whereas the decay of I Cl(Ca) occurred Figure 3. Calcium-induced calcium release is not required for muscarinic calcium release. (A) Block of ryanodine receptors with ruthenium red (50 M, dialyzed for 5 min) prevents caffeine (8 mm) induced calcium release and activation of I Cl(Ca), but has no effect on muscarinic (50 M mach) calcium release. (B) Release of calcium by caffeine (8 mm) prevents subsequent calcium release due to depletion of intracellular calcium stores. After washout of caffeine, mach evokes a typical response. Figure 4. Spontaneous transient inward currents contribute to rhythmic electrical activity. Spontaneous currents recorded in a nystatin voltage-clamped tracheal myocyte recorded in physiologic (potassium-containing) solutions. At potentials negative to 40 mv, transient inward chloride currents dominate electrical activity. At more positive potentials, transient outward potassium currents are observed.

4 S112 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 5. Time course of I Cl(Ca) and [Ca 2 ] i. The time course of [Ca 2 ] i increase (dots) and I Cl(Ca) (line) are shown following exposure to caffeine. The macroscopic current activates after [Ca 2 ] i increases to some threshold (horizontal arrow), but the current decays while [Ca 2 ] i remains elevated (vertical arrow). The current and [Ca 2 ] i increases have been scaled to equivalent maximums to show relative kinetics. The inset shows a similar experiment for the calcium-activated potassium current, where the current and [Ca 2 ] i signals overlap. substantially before the decline in spatially averaged [Ca 2 ] i. I Cl(Ca) began to decay before [Ca 2 ] i reached a peak value, and had declined to 50% of peak and completely decayed when [Ca 2 ] i had decreased by only 19% and 63% (from to and to nm), respectively. Whereas the activation rates of the [Ca 2 ] i and I Cl(Ca) were surprisingly similar given that the [Ca 2 ] i signal is a spatial average, there was always a dramatic discrepancy between decay rates of the two signals. The mean time required for the current to decay to 50% of peak (t 1/2 ) was , whereas the t 1/2 for the fall in [Ca 2 ] i was By contrast, similar experiments indicated that, following activation by caffeine, the calcium-activated potassium current (I K(Ca) ) decayed with kinetics that matched the cellular [Ca 2 ] i signal (inset, Figure 5). These results suggested that I Cl(Ca) inactivates by a mechanism independent of the removal of calcium. It was considered unlikely that the accelerated decay of I Cl(Ca) was due to a decline in near membrane [Ca 2 ] i not reflected in our spatially averaged [Ca 2 ] i measurement, since the decay of I K(Ca) closely matched this signal. Moreover, I Cl(Ca) but not I K(Ca) decayed in experiments in which ionomycin was used to achieve a sustained increase in [Ca 2 ] i. These experiments suggested that factors other than a decline in [Ca 2 ] i were associated with the decay of I Cl(Ca). To determine whether the inactivation of I Cl(Ca) was related to ATP hydrolysis, currents were examined in cells dialyzed with ATP, ADP, and the nonhydrolyzable analog AMP-PNP. In cells dialyzed with 1 mm ATP for 5 min using the standard whole-cell method, caffeine (8 mm) induced a similar calcium and current response as observed in nondialyzed cells (Figure 6A). However, as shown in Figure 6B (left), application of caffeine to cells dialyzed with 1 mm ADP (or AMP-PNP) for 5 min induced a current with a dramatically slowed rate of inactivation; moreover, the rate of current inactivation was quite similar to the rate of intracellular calcium decay. We therefore concluded that protein phosphorylation plays a role in the rapid inactivation of I Cl(Ca) observed under more physiologic conditions (nondialyzed cells). Figure 6. The rapid decay of I Cl(Ca) requires ATP. (A) Caffeine evokes a typical calcium and current response with the current decaying well before the fall in [Ca 2 ] i (inset). The cell was dialyzed with solution containing 1 mm ATP. (B) In a cell dialyzed with 1 mm ADP, I Cl(Ca) decays more slowly and the [Ca 2 ] i and current signals superimpose (inset). We reasoned that the increase in [Ca 2 ] i initiating I Cl(Ca) might activate calcium/calmodulin-dependent protein kinase II (CaMKII), and that activated CaMKII might promote channel closing by phosphorylating the channel protein or an associated protein, thereby constituting an efficient negative feedback system to ensure rapid termination of the postsynaptic response (24). To test this hypothesis, we dialyzed myocytes with 1 mm ATP plus either the calmodulin antagonist W7 or the CaMKII inhibitor KN93. The decay of I Cl(Ca) was slowed in cells dialyzed with either W7 (500 M) or KN93 (5 M), similar to the results obtained with AMP-PNP or ADP, and the CaMKII inhibitory peptide (residues ) also slowed the rate of current inactivation (Figure 7). Conversely H7, which has a relatively low affinity for CaMKII but inhibits other cellular kinases such as protein kinase C, cyclic AMP-dependent protein kinase, and cyclic GMP-dependent protein kinase, did not produce an appreciable effect on I Cl(Ca). These data further demonstrated that the inactivation of I Cl(Ca) is mediated by protein phosphorylation, and indicated that the signaling pathway involves CaMKII. CaMKII does not appear to play a role in the opening of Ca 2 -activated Cl channels, however, since neither the activation kinetics nor the peak amplitude of I Cl(Ca) were affected by intracellular dialysis of W7, KN93, or the regulatory peptide. We also examined whether CaMKII -mediated phosphorylation is sufficient to terminate I Cl(Ca) in the absence of a fall in [Ca 2 ] i, using ionomycin to induce a sustained increase in [Ca 2 ] i in cells dialyzed with ATP. Exposure to 10 M ionomycin resulted in a sustained increase in [Ca 2 ] i, but I Cl(Ca) still

5 Kotlikoff and Wang: Calcium Release in Airway Smooth Muscle Cells S113 Figure 7. Inhibition of CaMKII slows the decay of I Cl(Ca). The calcium/calmodulin-activated protein kinase II (CaMKII) peptide inhibitor (50 M, residues ) was dialyzed before caffeine stimulation. Current decay was markedly slowed by the kinase inhibitor. inactivated; in parallel experiments employing conditions designed to isolate the calcium-activated potassium current, that current was sustained following application of ionomycin. This experiment provides further evidence that the termination of I Cl(Ca) is not merely due to a more rapid decrease in near membrane [Ca 2 ] i than indicated by the spatially averaged calcium fluorescence signal, since the sustained elevation of the I K(Ca) indicates that the near-membrane calcium is maintained at near maximal levels. We therefore anticipated that a sustained increase in [Ca 2 ] i, together with inhibition of channel phosphorylation by CaMKII, would result in a sustained opening of Ca 2 -activated Cl channels. To test this hypothesis, myocytes were exposed to ionomycin under conditions that would effectively inhibit phosphorylation. As predicted, inhibition of phosphorylation by AMP-PNP, KN93, or the inhibitory peptide resulted in a sustained I Cl(Ca), whereas when cells were dialyzed with H7 the current was not sustained. Since phosphorylation of the channel or a related protein closed calcium-activated chloride channels, we sought to determine whether dephosphorylation was required for subsequent channel availability (i.e., whether phosphorylation results in channel inactivation). When cells were dialyzed with ATP, a second application of caffeine after 5 min reliably evoked calcium and current transients, although there was an approximately 10% decrease in the amplitude of the second I Cl(Ca), whereas the mean net increase in [Ca 2 ] i was not signif- Figure 8. The phosphorylation-dependent decay of I Cl(Ca) results from channel inactivation. (A) Consecutive exposure to caffeine resulted in equivalent increases in [Ca 2 ] i and only a slight decline in the magnitude of I Cl(Ca). (B) Substitution of ADP for ATP in the patch pipette results in the typical slowed rate of I Cl(Ca) decay but does not alter the current amplitude following a second exposure to caffeine, indicating that channels are available to open. ( C) Conversely, intracellular dialysis of ATP s results in a smaller initial I Cl(Ca) and virtually no response on the second application (despite the fact that the [Ca 2 ] i response was normal), indicating that calcium-activated chloride channels were not available to open. (D) Dialysis of okadaic acid also inhibited the initial caffeine response and almost abolished the subsequent response.

6 S114 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL icantly changed. Inhibition of phosphorylation by dialyzing cells with ADP or AMP-PNP also resulted in currents that could be evoked repeatedly with little decrease in net current, although the time course of current decay was slowed as previously shown, indicating that the calcium-dependent current decay does not reflect channel inactivation. In marked contrast to these results, however, when channel dephosphorylation was prevented either by thiophosphorylation or by inhibition of endogenous phosphatase activity, currents could not be subsequently activated. As shown in Figure 8, caffeine induced an I Cl(Ca) of smaller amplitude and faster decay in cells dialyzed with 1 mm ATP substituted for ATP, and a second application of caffeine (5 min after the first application) resulted in a dramatically decreased I Cl(Ca), which was reduced to 14 3% of the first response. Similarly, in cells dialyzed with the protein phosphatase-1 and -2A inhibitor, okadaic acid (500 nm), I Cl(Ca) was of smaller amplitude, and a second application of caffeine almost failed to evoke I Cl(Ca). In both experimental groups the magnitude or time course of the rise in [Ca 2 ] i was not different from control (ATP) for the first or second caffeine exposure. These results indicate that phosphorylation of the channel protein or a related protein induces the transition of calciumactivated chloride channels to an inactivated state and that dephosphorylation is required for subsequent channel availability. The gating behavior of Ca 2 -activated chloride channels can be described by a simplified model. Channels undergo a calcium-dependent transition from the closed to the open state, as indicated by the simple relationship between current and [Ca 2 ] i that obtains during the activation of I Cl(Ca) (1) and during its decay under conditions in which phosphorylation is inhibited. Under physiologic conditions open channels inactivate rapidly (before the fall in [Ca 2 ] i associated with a calcium release transient) in a process that is mediated by CaMKII, and transition from the inactivated state to the closed state requires protein dephosphorylation. These dual gating mechanisms represent a novel ion channel regulatory system. Inactivation of a calcium-dependent ion channel by a calcium-dependent kinase comprises an efficient negative feedback mechanism that uncouples current from the [Ca 2 ] i signal, ensuring rapid termination of the depolarizing stimulus. SUMMARY These studies have revealed an unexpected level of complexity in the regulation of calcium-activated chloride channels in airway smooth muscle cells, and the importance of intracellular release events in determining the electrical activity of airway smooth muscle. 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