Differential regulation of Ca2+ sparks and Ca2+ waves by UTP in rat cerebral artery smooth muscle cells

Jonathan H. Jaggar and Mark T. Nelson

Department of Pharmacology, the University of Vermont, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activates phospholipase C, shifted Ca2+ signaling from sparks to waves in the smooth muscle cells of rat cerebral arteries. UTP decreased the frequency of Ca2+ sparks and transient Ca2+-activated K+ (KCa) currents and increased the frequency of Ca2+ waves. The UTP-induced reduction in Ca2+ spark frequency did not reflect a decrease in global cytoplasmic Ca2+, Ca2+ influx through voltage-dependent Ca2+ channels (VDCC), or Ca2+ load of the sarcoplasmic reticulum (SR), since global Ca2+ was elevated, blocking VDCC did not prevent the effect, and SR Ca2+ load did not decrease. However, blocking protein kinase C (PKC) with bisindolylmaleimide I did prevent UTP reduction of Ca2+ sparks and transient KCa currents. UTP decreased the effectiveness of caffeine, which increases the Ca2+ sensitivity of ryanodine-sensitive Ca2+ release (RyR) channels, to activate transient KCa currents. This work supports the concept that vasoconstrictors shift Ca2+ signaling modalities from Ca2+ sparks to Ca2+ waves through the concerted actions of PKC on the Ca2+ sensitivity of RyR channels, which cause Ca2+ sparks, and of inositol trisphosphate (IP3) on IP3 receptors to generate Ca2+ waves.

ryanodine receptor; calcium channel; calcium-sensitive potassium channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTRACELLULAR CALCIUM IONS (Ca2+) regulate a variety of cellular processes, ranging from cellular contraction to gene expression (for review see Ref. 6). Recently, the view that the global intracellular Ca2+ concentration ([Ca2+]i) is homogeneously distributed in cells has been altered by the discovery of local and global subcellular Ca2+ signaling events (for reviews see Refs. 2, 6, 18). Frequency and amplitude modulation of these events, in addition to the subcellular localization of the Ca2+ signal, can lead to the regulation of discrete Ca2+-sensitive cellular processes (2). In arterial smooth muscle, two distinct local Ca2+ signaling events have been described (termed "Ca2+ sparks" and "Ca2+ waves"), which differ with respect to temporal kinetics and spatial localization. Interestingly, Ca2+ sparks and Ca2+ waves have also been proposed to have opposing effects on arterial contractility (16, 30).

Ca2+ sparks have been characterized in cardiac (5), skeletal (22, 43), and smooth muscle cells (30) and occur due to the opening of a number of sarcoplasmic reticulum (SR) ryanodine-sensitive Ca2+-release (RyR) channels, resulting in a rapid, highly localized increase in [Ca2+]i. Recent estimates have suggested that the local change in Ca2+ concentration ([Ca2+]) by a single Ca2+ spark may be in the order of 10-100 µM (32). However, in arterial smooth muscle the impact of Ca2+ sparks on the global [Ca2+]i is small (<2 nM), since Ca2+ sparks occur at a rate of about 1 per second per cell, and the Ca2+ from a spark spreads into only ~1% of the cell volume (19, 30). In arterial smooth muscle, the majority of Ca2+ sparks occur very close to the sarcolemma and activate a number of Ca2+-sensitive potassium (KCa) channels (30, 32) [previously referred to as a spontaneous transient outward current or "STOC" (1)]. A single Ca2+ spark is capable of inducing a significant membrane potential hyperpolarization [~20 mV (7, 18)] and, when averaged across the arterial wall, a tonic hyperpolarization of the smooth muscle membrane potential (18, 23, 30). Inhibition of Ca2+ sparks leads to membrane potential depolarization, activation of voltage-dependent Ca2+ channels, an increase in the [Ca2+]i, and vasoconstriction (23, 30). Spontaneous transient inward currents ("STICs"), which occur due to the activation of a number of Ca2+-activated Cl- (ClCa) channels have been described in some types of smooth muscle (for review see Ref. 25). In guinea pig tracheal myocytes, Ca2+ sparks have been described to activate STICs (48), and a single Ca2+ spark can activate both KCa and ClCa channels, which results in an outward KCa current followed by an inward ClCa current (termed a "STOIC") (48).

In contrast to Ca2+ sparks, Ca2+ waves are propagating elevations in [Ca2+]i that have been implicated as mediators of constriction (cf. Ref. 16). The frequency of Ca2+ waves increases in arterial smooth muscle cells when vasoconstrictors that elevate cytoplasmic levels of inositol trisphosphate (IP3) are applied, indicating that Ca2+ release through IP3 receptors contributes to Ca2+ waves (16, 26). RyR channels may also contribute to Ca2+ waves, since IP3-mediated Ca2+ release can activate RyR channels (3).

We sought to determine the mechanisms by which vasoconstrictors modify Ca2+ signaling pathways in arterial smooth muscle. In general, vasoconstrictors stimulate phospholipase C (PLC) through activation of an associated G protein (Gq). PLC cleaves phosphatidylinositol bisphosphate to diacylglycerol (DAG) and IP3. Each of these second messengers will have distinct effects in the cell. DAG activates protein kinase C (PKC) and IP3 activates IP3 receptors in the SR membrane, leading to Ca2+ release. We have previously shown that activators of PKC decrease the frequency of Ca2+ sparks in isolated cerebral artery smooth muscle cells, which may occur through a direct effect of PKC on the RyR channel (4). The actions of vasoconstrictors will not only involve actions of PKC but will also be mediated through increases in the cytoplasmic IP3 and intracellular Ca2+ concentrations. Activation of IP3 receptors could have opposing effects on RyR channels by increasing cytoplasmic [Ca2+], which will activate RyR channels (19, 30), and by decreasing SR Ca2+ load, which will inhibit RyR channels (49). Therefore, the impact of vasoconstrictors on Ca2+ sparks is unclear.

We tested the hypothesis that the concerted actions of signal transduction pathways from vasoconstrictors leads to a decrease in Ca2+ sparks and an elevation of Ca2+ waves. Uridine 5'-triphosphate (UTP), a potent vasoconstrictor of cerebral arteries (44), binds to P2Y receptors linked to PLC in smooth muscle cells (12, 39, 40). UTP-induced changes in subcellular Ca2+ signaling (Ca2+ sparks and Ca2+ waves) were measured in the smooth muscle cells of intact endothelium-denuded cerebral arteries, using a laser scanning confocal microscope and the fluorescent Ca2+ dye fluo 3. The effects of UTP on transient KCa currents in isolated voltage-clamped smooth muscle cells were examined. SR Ca2+ load was monitored by applying a high concentration of caffeine (10 mM) to induce the rapid release of stored Ca2+. Application of UTP to intact, endothelium-denuded cerebral arteries decreased the frequency of Ca2+ sparks and increased the frequency of Ca2+ waves in smooth muscle cells. Furthermore, UTP decreased the frequency and amplitude of transient KCa currents in isolated voltage-clamped myocytes. Inhibition of dilatory Ca2+ signaling (Ca2+ sparks) and activation of contractile signaling (Ca2+ waves) represents a potential novel mechanism of action for vasoconstrictors.

Thus the vasoconstrictor UTP shifts Ca2+ signaling modalities from Ca2+ sparks to Ca2+ waves through the concerted actions of PKC and IP3.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Sprague-Dawley rats (12-14 wk) of either sex were euthanized by peritoneal injection of pentobarbital solution (150 mg/kg). The brain was removed and placed into ice-cold, oxygenated (95% O2-5% CO2), physiological bicarbonate solution (PBS) containing (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11 glucose (pH 7.4). Posterior cerebral and cerebellar arteries were removed and were cleaned of basolateral connective tissue. For experiments using intact arteries, endothelium was removed by allowing an air bubble to remain in the lumen of the artery for 2 min, followed by a 30-s wash with H2O (45).

Confocal calcium measurements. To provide a planar face for imaging, endothelium-denuded cerebral arteries were slipped over rectangular glass cannullae (220 µm × 40 µm × 10 mm), whose ends were fire polished to reduce luminal arterial damage (see Ref. 19). Arteries were placed into a physiological K+ solution (PSS) of the following composition (in mM): 136 NaCl, 6 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, and 10 glucose (pH 7.4 with NaOH), containing 10 µM fluo 3-AM (Molecular Probes) and 0.05% Pluronic acid (Molecular Probes) for 60 min at 22°C. To allow fluo 3 deesterification, arteries were then placed in PSS for 30 min at 22°C. Arteries were imaged using a Noran Oz laser scanning confocal microscope and a ×60 water-immersion lens (numerical aperture = 1.2) by illuminating with a krypton/argon laser at 488 nm. Images of the vessel wall (56.3 µm × 52.8 µm or 256 pixels × 240 pixels) were recorded every 16.7 ms (60 images/s). Under each condition, two different representative areas of the same artery were each scanned for 10 s. The same area of artery was not scanned more than once, to avoid any laser-induced changes in Ca2+ signaling, and effects of substances were measured in paired experiments. To allow comparison with the single-cell patch-clamp data, an elevated K+ (30 mM extracellular K+) bath solution that contained (in mM) 112 NaCl, 30 KCl 10 HEPES, 2 CaCl2, 1 MgCl2, and 10 glucose (adjusted to pH 7.4 with NaOH) was used to depolarize the smooth muscle cells of intact arteries to approximately -40 mV. Elevating extracellular K+ from 6 to 30 mM increases the frequency of Ca2+ sparks 4.3-fold and increases [Ca2+]i 1.7-fold (19). Files were stored on compact disc for future analysis.

Patch-clamp electrophysiology. Individual smooth muscle cells were enzymatically dissociated from cerebral arteries in a manner similar to that previously described (36). K+ currents were measured using the whole cell, perforated-patch configuration (14) of the patch-clamp technique (11), using an Axopatch 200A amplifier (Axon Instruments, Foster City CA). Bathing (6 K+) solution was PSS (composition described previously). The pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (pH 7.2 with KOH). Membrane currents were recorded (sample rate 2 kHz; filtered at 500 Hz) at -40 mV. To determine the mean amplitude and frequency of transient KCa currents, analysis was performed off-line using a custom analysis program. The threshold of detection for these events was set at 2.5 times the single-channel amplitude at -40 mV or at 4.9 pA. In the presence of ryanodine or thapsigargin, the simultaneous opening of three single KCa channels was not observed at -40 mV (4, 30, 34).

Conventional Ca2+ imaging. Isolated smooth muscle cells were incubated with 0.25 µM fura 2-AM for 15 min. Cells were then washed and allowed to sit in the dark for 20 min to allow dye deesterification before measurements were made. Ca2+ was measured ratiometrically (340:380 nm) using IMAGE-1/FL quantitative fluorescence measurement software (Universal Imaging, West Chester, PA). Fluorescence ratios were converted to Ca2+ concentrations (as described in Ref. 10), using an apparent Kd of Ca2+ for fura 2 of 282 nM (23).

Ca2+ spark, Ca2+ wave, and global F/F0 analysis. Ca2+ sparks were detected using custom analysis software written in our laboratory by Dr. Adrian Bonev (using IDL 5.0.2; Research Systems, Boulder, CO). Detection of Ca2+ sparks was performed by dividing an area 1.54 µm (7 pixels) × 1.54 µm (7 pixels) (i.e., 2.37 µm2) in each image (F) by a baseline (F0) that was determined by averaging 10 images without Ca2+ spark activity. Ca2+ spark frequency in intact arteries, under a given condition, was determined by measuring the number of sparks that occurred in two 56.3 µm × 52.8 µm areas (~20 cells) each scanned for 10 s. Mean data for each condition is the subsequent average ± SE of these artery frequency values. Ca2+ spark amplitude was calculated as F/F0. Ca2+ spark and Ca2+ wave activity were measured on the same cells. Ca2+ waves were detected by eye by placing 2.2 µm × 2.2 µm (10 × 10 pixels) boxes in individual myocytes, and referring to a change in F/F0 >1.3 that remained elevated for >200 ms. Global F/F0 for a given condition was calculated as the mean pixel value of 60 different images acquired over 10 s. Changes in global F/F0 under certain conditions were determined in paired arteries.

Statistical analysis. Values are expressed as means ± SE. Paired and unpaired Student's t-tests were performed where appropriate, with P < 0.05 considered significant.

Chemicals. Unless otherwise stated, all chemicals used in this study were obtained from Sigma Chemical (St. Louis, MO). Bisindolylmaleimide I (Bis I) was purchased from Calbiochem (La Jolla, CA). All experiments were conducted at room temperature (20-22°C).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UTP decreases Ca2+ spark frequency in the smooth muscle cells of intact cerebral arteries. The effects of UTP on Ca2+ sparks were examined in intact cerebral arteries using a laser scanning confocal microscope and the fluorescent Ca2+ dye fluo 3. The bathing solution contained 30 mM K+ to depolarize the smooth muscle cells to approximately -40 mV, a membrane potential similar to that observed in cerebral arteries pressurized to 60 mmHg (23, 30). Arteries were endothelium denuded (for procedure see METHODS) to avoid any effects of endothelium-derived relaxing factors (29, 47) on Ca2+ signaling mechanisms in the smooth muscle cells. Figure 1A indicates the average fluorescence over 10 s (60 images averaged) in 56.3 µm × 52.8 µm (256 pixels × 240 pixels) areas of the same rat cerebral artery in control (30 mM K+) and with 30 µM UTP (top). White boxes (1.54 µm × 1.54 µm or 7 × 7 pixels) indicate the locations of detected Ca2+ sparks, with numbered boxes illustrating the corresponding changes in fractional fluorescence (F/F0) for four such events below each respective image.


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Fig. 1.   UTP decreases Ca2+ spark frequency in smooth muscle cells of intact cerebral arteries. A: average fluorescence over 10 s (60 images averaged) in 56.3 µm × 52.8 µm (256 pixels × 240 pixels) areas of the same rat cerebral artery in control (30 mM K+) and with 30 µM UTP. Ca2+ sparks were detected using a custom analysis program written in our laboratory using IDL (for detection criteria see METHODS). Labeled boxes [1.54 µm × 1.54 µm (7 × 7 pixels)] indicate the locations of detected events, with numbered boxes illustrating 4 such events represented below each respective image. Traces represent F/F0 changes with time. B: mean effects of 10 and 30 µM UTP on Ca2+ spark frequency. UTP (10 µM) reduced Ca2+ spark frequency (control, 9 arteries, 18 sections, 180 s total) from 1.80 ± 0.42 to 0.68 ± 0.45 Hz, or by 56.5 ± 18.6% (n = 4 paired arteries, 8 sections, 80 s total), and 30 µM UTP from 2.07 ± 0.22 to 0.31 ± 0.04 Hz, or by 84.3 ± 1.9% (n = 5 paired arteries, 10 sections, 100 s total).

UTP decreased the frequency of Ca2+ sparks in the smooth muscle cells in a concentration-dependent manner (Fig. 1B). UTP at 10 and 30 µM reduced Ca2+ spark frequency by 56.5 ± 18.6% (n = 4 paired arteries) and by 84.3 ± 1.9% (n = 5 paired arteries), respectively (in a 56.3 µm × 52.8 µm scan area, Fig. 1, A and B). The amplitude of Ca2+ sparks, as measured by F/F0, was not altered by UTP [for 10 µM UTP: control 1.72 ± 0.03 (n = 98), 10 µM UTP 1.69 ± 0.04 (n = 27); for 30 µM UTP: control 1.79 ± 0.02 (n = 228), 30 µM UTP 1.75 ± 0.03 (n = 53)].

Ca2+ sparks were not observed in the absence or presence of UTP (30 µM) in myocytes of intact cerebral arteries (n = 6 arteries) that had been pretreated with ryanodine (10 µM, 15 min), suggesting that all Ca2+ sparks were due to Ca2+ release through RyR channels in the SR membrane. These results demonstrate that UTP decreases Ca2+ spark frequency in the smooth muscle cells of intact cerebral arteries.

UTP increases the frequency of Ca2+ waves in the smooth muscle cells of intact cerebral arteries. Vasoconstrictors have been shown to increase the frequency of Ca2+ waves in myocytes of rat tail artery (16) and to increase the frequency of Ca2+ oscillations in rat mesenteric artery myocytes (26). In contrast to Ca2+ sparks, which are rapid, highly localized intracellular Ca2+ transients, Ca2+ waves are propagating global Ca2+ events. Figure 2A illustrates the same 56.3 µm × 52.8 µm images (top) illustrated in Fig. 1A.


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Fig. 2.   UTP increases Ca2+ wave frequency in cerebral artery smooth muscle cells. A: top panels represent the same 56.3 µm × 52.8 µm images shown in Fig. 1A. Boxes [2.2 µm × 2.2 µm (10 × 10 pixels)] indicate locations where global changes in F/F0 over 10 s were measured in myocytes. F0 was determined by averaging the first 10 images of the 10-s acquisition period (600 images). In control conditions (30 mM K+) 2 Ca2+ waves were observed in 10 cells, whereas, with 30 µM UTP, 10 Ca2+ waves occurred in 12 cells. B: propagation of a Ca2+ wave in a myocyte of an intact cerebral artery in the presence of UTP (30 µM). Panel represents the same 56.3 µm × 52.8 µm image shown in Figs. 1A and 2A of an intact cerebral artery in the presence of UTP (30 µM). Boxes [2.2 µm × 2.2 µm (10 pixels × 10 pixels)] placed 13.3 µm apart indicate the locations where global changes in F/F0 over 10 s were measured in the same arterial myocyte. Colored traces to the right of the image indicate the change in F/F0 for the respective colored box in the image. The Ca2+ wave propagated from right to left in the image.

Under control conditions, Ca2+ waves were observed, on average, in 11.2 ± 4.6% of myocytes (n = 176 cells; 9 arteries; Figs. 2A and 3). UTP at 10 and 30 µM increased the frequency of Ca2+ waves to 36.3 ± 14.4% of myocytes (n = 81 cells; 4 paired arteries) and 78.7 ± 13.3% of myocytes (n = 118 cells; 5 paired arteries), respectively (Fig. 3). The mean propagation velocity of Ca2+ waves in control (10 ± 3 µm/s; range = 7-13 µm/s, n = 5 waves) was elevated by UTP (30 ± 20 µm/s; range = 10-59 µm/s, n = 10 waves). The average fractional fluorescence (F/F0) of the acquisition area (over 10 s) was significantly increased 1.35 ± 0.15-fold by UTP (30 µM; P < 0.05, n = 5 paired arteries), which may represent the additive effects of elevated Ca2+ wave frequency and increased global [Ca2+]i.


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Fig. 3.   Mean incidence of Ca2+ waves in the smooth muscle cells of intact cerebral arteries. Application of 10 µM and 30 µM UTP increased the incidence of Ca2+ waves from 11.2 ± 4.6% of myocytes (n = 9 arteries) to 36.3 ± 14.4% (n = 4 paired arteries) and 78.7 ± 13.3% of myocytes (n = 5 paired arteries), respectively.

Ca2+ waves could arise from the activation of RyR channels and IP3 receptors. To test the role of RyR channels in Ca2+ waves, arteries were pretreated with ryanodine (10 µM, 15 min) to block RyR channels. Ca2+ waves were not observed in the smooth muscle cells in control or after application of UTP (30 µM; n = 6 arteries). These results indicate that UTP increases the frequency of Ca2+ waves in the smooth muscle cells of intact cerebral arteries.

UTP inhibits Ca2+ sparks and activates Ca2+ waves through activation of PLC. UTP binds to P2Y receptors linked to PLC in smooth muscle cells (12, 39, 40). To determine the role of PLC in UTP regulation of Ca2+ signaling, we pretreated arteries with U-73122 (2 µM), an inhibitor of PLC (15-min incubation). UTP (30 µM) did not significantly change the frequency of Ca2+ sparks in the smooth muscle cells of arteries that had been pretreated with U-73122 (control, 2.85 ± 0.89 Hz; 30 µM UTP, 2.2 ± 1.05 Hz or 80.4 ± 18.9% of control; n = 4 paired arteries). Similarly, UTP did not significantly change the frequency of Ca2+ waves (control, 23.4 ± 11.1%, n = 94 cells; 30 µM UTP, 36.0 ± 10.6%, n = 91 cells; n = 4 paired arteries). These results demonstrate that UTP-mediated Ca2+ spark inhibition and Ca2+ wave activation occur through activation of PLC.

UTP inhibits transient KCa currents in isolated cerebral artery smooth muscle cells. Ca2+ sparks activate nearby plasmalemmal KCa channels to elicit transient KCa currents (30, 32). If UTP decreases Ca2+ spark frequency, then it should also decrease the frequency of transient KCa currents. In isolated voltage-clamped (-40 mV) cerebral artery myocytes, UTP at 10 and 100 µM decreased transient KCa current frequency by 65.8 ± 12.7% (n = 5) and by 98.9 ± 0.6% (n = 3), respectively (Fig. 4, A and B). UTP (10 and 100 µM) reduced mean transient KCa current amplitude by 43.0 ± 12.7% (n = 5) and by 52.6 ± 20.6% (n = 3), respectively (Fig. 4, A and B). Inhibition of transient KCa currents was sustained throughout UTP application (at least 10 min) and was readily reversible on washout (Fig. 4A). Figure 4A also illustrates the effect of applying UTP (1 µM), which reduced transient KCa current frequency from 2.7 Hz (control) to 2.0 Hz (by 26.6%), and mean amplitude from 19.0 to 14.7 pA (by 25.4%). In addition, Fig. 4A illustrates that UTP (1-100 µM) significantly reduced the mean outward K+ current (white line).


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Fig. 4.   UTP decreases transient Ca2+-activated K+ (KCa) current frequency and amplitude in isolated, voltage-clamped cerebral artery myocytes. A: original record of transient KCa currents recorded in an isolated smooth muscle cell from a rat cerebral artery. Holding potential was -40 mV. UTP (1-100 µM) decreased the frequency and amplitude of transient KCa currents and the mean outward K+ current (white line, obtained by filtering the raw data trace at 2 Hz, and subsequently averaging 200 adjacent data points) in a concentration-dependent manner. B: average changes in transient KCa current frequency and amplitude relative to the pretreatment control levels with 10 µM (n = 5) and 100 µM UTP (n = 3). UTP (10 µM) decreased transient KCa current frequency from 1.48 ± 0.3 to 0.59 ± 0.28 Hz, or by 65.8 ± 12.7%, and mean amplitude from 18.2 ± 3.2 to 8.8 ± 1.1 pA, or by 43.0 ± 12.7%. UTP (100 µM) decreased transient KCa current frequency from 2.07 ± 0.37 to 0.02 ± 0.01 Hz, or by 98.9 ± 0.6%, and mean transient KCa current amplitude from 14.4 ± 5.7 to 6.2 ± 0.6 pA, or by 52.6 ± 20.6%. *Significant decrease in transient KCa current frequency or amplitude compared with control (P < 0.05).

UTP does not reduce caffeine-induced Ca2+ transients. UTP could conceivably decrease Ca2+ spark frequency by reducing SR Ca2+ load (49). To test this possibility, global [Ca2+]i was measured in isolated cerebral artery myocytes using a conventional Ca2+ imaging system and the ratiometric Ca2+ indicator fura 2. To determine SR Ca2+ load, caffeine (10 mM), which increases the affinity of the Ca2+ activation site on the RyR channel for Ca2+ (33), was applied to isolated cerebral artery myocytes, to maximally activate RyR channels in the SR and rapidly deplete the SR (see Ref. 4 for similar protocol). Caffeine-induced cytoplasmic elevations of [Ca2+]i under control conditions were compared with those obtained 10 min after application of UTP (10 µM). Application of UTP induced repetitive Ca2+ transients (Fig. 5) that may represent the Ca2+ waves we observed in intact arteries (Fig. 2, A and B). The mean amplitude of UTP-induced Ca2+ transients was 249 ± 11 nM Ca2+ (n = 34 transients). Mean caffeine-induced Ca2+ transients were 402 ± 12 nM in control and 402 ± 17 nM with 10 µM UTP (n = 14). These results indicate that the Ca2+ load of the SR is not decreased by UTP, suggesting that inhibition of Ca2+ sparks does not occur through this mechanism.


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Fig. 5.   UTP does not reduce caffeine-induced Ca2+ transients. Transient increases in cytosolic Ca2+ ([Ca2+]cyt ) in response to 10 mM caffeine (caff) before and after application of UTP (10 µM). The black line represents the mean [Ca2+]cyt of 10 isolated cerebral artery smooth muscle cells. Gray inset shows oscillations of [Ca2+]cyt that occurred with UTP (10 µM) in a single cell (oscillations occurred in all cells but the amplitude of these events is reduced through averaging). Cells were loaded with the Ca2+ indicator fura 2-AM and Ca2+ measured ratiometrically (340:380 nm) using IMAGE-1/FL quantitative fluorescence equipment.

UTP does not decrease, but increases Ca2+ spark frequency and transient KCa current frequency in the presence of Bis I, a blocker of PKC. Activators of PKC (phorbol ester and dioctanoyl-sn-glycerol) have been shown to inhibit Ca2+ sparks and may act through a direct PKC effect on the RyR channel (4). Because UTP leads to PKC activation, UTP could inhibit Ca2+ sparks and transient KCa currents through a PKC-induced effect on the RyR channel. We explored this possibility by assessing the effects of UTP on intact cerebral arteries (Ca2+ spark measurements) and isolated cerebral artery myocytes (transient KCa current measurements), which had been pretreated with Bis I (1 µM, 30 min) to inhibit PKC (42).

UTP (30 µM) did not decrease but significantly increased the frequency of Ca2+ sparks in myocytes of intact cerebral arteries pretreated with Bis I (1 µM), from 1.43 ± 0.36 to 2.08 ± 0.34 Hz, or to 154.8 ± 18.7% of control (n = 4 paired arteries; P < 0.05; Fig. 6A). The mean amplitude (F/F0) of Ca2+ sparks was not changed with 30 µM UTP (control, 1.64 ± 0.02, n = 115; 30 µM UTP, 1.64 ± 0.02, n = 166). Bis I did not affect UTP elevation of Ca2+ waves. In the same arteries pretreated with Bis I, UTP (30 µM) increased the frequency of Ca2+ waves to 77.3 ± 4.5% of myocytes (n = 4 paired arteries), similar to that observed in control arteries. These results suggest that UTP inhibition of Ca2+ sparks is mediated through activation of PKC, and UTP activation of Ca2+ waves occurs independently of PKC.


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Fig. 6.   UTP increases the frequency of Ca2+ sparks and transient KCa currents when applied in the presence of bisindolylmaleimide I (Bis I), a potent blocker of protein kinase C. A: UTP (30 µM) significantly increased the frequency of Ca2+ sparks in myocytes of intact cerebral arteries pretreated (30 min) with Bis I (1 µM), from 1.43 ± 0.36 to 2.08 ± 0.34 Hz, or to 154.8 ± 18.7% of control (n = 4 paired arteries; P < 0.05). B: UTP increased transient KCa current frequency in isolated voltage-clamped smooth muscle cells pretreated with Bis I (1 µM). In this experiment transient KCa current frequency and mean amplitude were: control, 2.93 Hz and 27.7 pA; 30 µM UTP, 3.83 Hz and 25.9 pA; wash, 3.13 Hz and 23.7 pA, respectively. C: average changes in transient KCa current frequency and amplitude in isolated voltage-clamped (-40 mV) smooth muscle cells (n = 5). Similar to the effects of UTP on Ca2+ sparks, UTP (30 µM) significantly increased transient KCa current frequency in cells pretreated with 1 µM Bis I from 4.5 ± 0.98 to 5.9 ± 1.0 Hz, or by 139.9 ± 20.7% (n = 5; P < 0.05), at -40 mV. The amplitude of transient KCa currents was unaffected by UTP in Bis I-pretreated cells (control 29.6 ± 5.3 pA; 30 µM UTP 27.6 ± 4.6 pA; or 94.2 ± 4.3% of control; n = 5; C). *Significant change in Ca2+ spark frequency or transient KCa current frequency compared with control (P < 0.05).

If block of PKC prevents UTP inhibition of Ca2+ sparks, then it should also prevent UTP inhibition of transient KCa currents. To test this, isolated cerebral artery smooth muscle cells were incubated with Bis I (1 µM) at room temperature for 30 min before patch formation. Bis I (1 µM ) alone had no effect on transient KCa current frequency (control, 1.88 ± 0.48 Hz; 1 µM Bis I, 2.10 ± 0.57 Hz; or 110.9 ± 16.6% of control) or amplitude (control, 16.4 ± 1.8 pA; 1 µM Bis I, 16.9 ± 1.3 pA; or 106.6 ± 12.9% of control) at -40 mV (n = 5). Similar to the effects of UTP on Ca2+ sparks, UTP (30 µM) significantly increased transient KCa current frequency in cells pretreated with 1 µM Bis I by 139.9 ± 20.7% (P < 0.05) at -40 mV (Fig. 6, B and C). The amplitude of transient KCa currents was unaffected by UTP (30 µM) in Bis I-pretreated cells (94.2 ± 4.3% of control; n = 5; Fig. 6C). These results indicate that UTP inhibits Ca2+ sparks and associated transient KCa currents through activation of PKC. Furthermore, if PKC is blocked, UTP activates Ca2+ sparks.

UTP inhibits transient KCa currents in the presence of Cd2+, a blocker of voltage-dependent Ca2+ channels. Activation of voltage-dependent Ca2+ channels, through membrane potential depolarization or application of BAY K 8644, increases the frequency of Ca2+ sparks in cerebral artery smooth muscle cells (19, 30). Furthermore, block of voltage-dependent Ca2+ channels reduces the frequency of Ca2+ sparks (19) and transient KCa currents (4). Therefore, UTP could conceivably inhibit Ca2+ sparks and transient KCa currents through a PKC-induced inhibition of voltage-dependent Ca2+ channels. To examine this possibility, the effects of UTP (30 µM) were tested in the presence of the inorganic Ca2+ channel blocker Cd2+ (250 µM) in voltage-clamped (-40 mV) cerebral artery myocytes (15). Cd2+ (250 µM) significantly reduced the mean frequency of transient KCa currents by 54.5 ± 6.6% (P < 0.05) but did not change mean amplitude (n = 7, Fig. 7, A-C). In the presence of Cd2+ (250 µM), UTP (30 µM) reduced transient KCa current frequency by 77.9 ± 6.7% (Fig. 7, A and B; P < 0.05) and mean transient KCa current amplitude by 57.2 ± 11.2% (n = 7; P < 0.05; Fig. 7, A-C). These results demonstrate that UTP inhibits transient KCa currents independently of changes in Ca2+ channel activity.


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Fig. 7.   UTP inhibits transient KCa currents when applied in the presence of 250 µM Cd2+, a blocker of voltage-dependent channels. A: original record illustrating transient KCa current activity in an isolated voltage-clamped cerebral artery smooth muscle cell (-40 mV). In control, transient KCa currents, frequency was 1.52 Hz and mean amplitude was 29.2 pA. After application of 250 µM Cd2+, frequency and amplitude were 0.73 Hz and 22.8 pA, respectively, and with 30 µM UTP + 250 µM Cd2+ were 0.25 Hz and 18.0 pA, respectively. B: mean transient KCa current frequencies from 7 cells. Cd2+ (250 µM) significantly reduced transient KCa current frequency from 1.51 ± 0.29 to 0.62 ± 0.24 Hz, or by 54.5 ± 6.6% (P < 0.05). UTP (30 µM) applied in the presence of 250 µM Cd2+ further reduced transient KCa current frequency to 0.11 ± 0.04 Hz, or by 77.9 ± 6.7% (P < 0.05). C: mean transient KCa current amplitude was unaffected by 250 µM Cd2+ (control, 31.3 ± 7.2 pA; 250 µM Cd2+ 32.7 ± 9.2 pA, n = 7), whereas 30 µM UTP applied in the presence of Cd2+ significantly reduced transient KCa current amplitude to 11.8 ± 3.1 pA, or by 57.2 ± 11.2% (n = 7, P < 0.05).

Caffeine reverses UTP-induced inhibition of transient KCa currents. Cytoplasmic Ca2+ is the key regulator of RyR channel activity. It is conceivable that UTP decreases Ca2+ spark frequency through a PKC-induced reduction in the Ca2+ sensitivity of the RyR channel. To test this possibility, we determined the effectiveness of caffeine to activate transient KCa currents in the absence and presence of UTP. Caffeine activates RyR channels by increasing the affinity of the Ca2+ activation site for Ca2+ (33). At concentrations <1 mM, caffeine has been demonstrated to increase the frequency of Ca2+ sparks in isolated smooth muscle cells from cerebral artery (9), portal vein (27), and airway (38). If PKC inhibits Ca2+ sparks through decreasing RyR channel Ca2+ sensitivity, a number of testable predictions exist: 1) caffeine should reverse UTP-induced Ca2+ spark inhibition, and 2) UTP should decrease caffeine activation of Ca2+ sparks, i.e., increased concentrations of caffeine should be required to activate Ca2+ sparks when applied in the presence of UTP.

First, the concentrations of caffeine that would induce stable activation of transient KCa currents in voltage-clamped cerebral artery myocytes were determined. Caffeine (25 µM) significantly increased the frequency of transient KCa currents by 188.2 ± 12.6% (n = 5; P < 0.05, Fig. 8, A and B), whereas the mean amplitude of transient KCa currents did not change (n = 5, Fig. 8B). Transient KCa current activation was maintained throughout application of caffeine (25 µM), and mean amplitude did not change, suggesting that SR Ca2+ load did not decrease (49). In contrast, when applied in the presence of UTP (30 µM), caffeine (25 µM) did not change the frequency of transient KCa currents (n = 4, Fig. 8, A and C).


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Fig. 8.   Effectiveness of caffeine activation of transient KCa currents is reduced by UTP. A: original records illustrating the effects of 25 µM caffeine and 500 µM caffeine on transient KCa currents, in the presence and absence of 30 µM UTP, in isolated voltage-clamped (-40 mV) cerebral artery smooth muscle cells. Top left trace: effect of 25 µM caffeine, which induced a reversible increase in transient KCa current frequency. Top right trace: effect of applying 25 µM caffeine in the presence of 30 µM UTP, which did not change transient KCa current frequency. Caffeine (500 µM; bottom left trace) induced a large transient K+ current (mean = 602.8 ± 180.4 pA, n = 4 cells), probably due to the simultaneous activation of large numbers of ryanodine-sensitive Ca2+-release (RyR) channels, causing release of Ca2+ from the sarcoplasmic reticulum and subsequent activation of many KCa channels. When applied in the presence of 30 µM UTP, 500 µM caffeine (bottom right trace) did not induce a large transient K+ current and was an effective activator of transient KCa currents. B: average values from 5 cells illustrating the effects of 25 µM caffeine, which significantly increased transient KCa current frequency from 0.72 ± 0.22 to 1.34 ± 0.43 Hz (n = 5; P < 0.05), or to 188.1 ± 12.6% of control, whereas amplitude was unaffected (control, 16.2 ± 4.4 pA; 25 µM caffeine, 17.3 ± 1.9 pA, or 122.2 ± 16.8% of control). C: average effects of 25 and 500 µM caffeine on transient KCa current frequency when applied in the presence of 30 µM UTP. Caffeine (25 µM) was ineffective when applied in the presence of 30 µM UTP (control, 1.76 ± 0.94 Hz; 30 µM UTP, 0.14 ± 0.10 Hz; 25 µM caffeine + 30 µM UTP, 0.14 ± 0.11 Hz; n = 4). However, 500 µM caffeine significantly increased transient KCa current frequency from 5.1 ± 3.2% of control to 35.1 ± 11.7% of control (control, 1.33 ± 0.40 Hz; 30 µM UTP, 0.04 ± 0.02 Hz; 500 µM caffeine + 30 µM UTP, 0.36 ± 0.31 Hz; n = 6; P < 0.05). *Significant change in transient KCa current frequency compared with control (P < 0.05).

UTP reduced caffeine activation of transient KCa currents even at a much higher concentration. When applied to myocytes in the absence of UTP, 500 µM caffeine induced a large transient outward K+ current (Fig. 8A, mean = 602.8 ± 180.4 pA, n = 4 cells), which would correspond to the activation of >300 KCa channels. The caffeine-induced K+ transient was probably due to the simultaneous activation of a large number of RyR channels that would significantly raise [Ca2+]i (e.g., see Ref. 21). However, when applied in the presence of UTP (30 µM), 500 µM caffeine did not induce a large transient outward K+ current, but significantly increased the frequency of transient KCa currents from 5.1 ± 3.2% to 35.1 ± 11.7% of the value measured in the absence of UTP (n = 6, P < 0.05, Fig. 8, A and C).

These results indicate that UTP reduced caffeine activation of Ca2+ sparks and suggest that UTP reduces the Ca2+ sensitivity of RyR channels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We provide the first evidence that UTP, a potent vasoconstrictor (44) that activates PLC (12, 39, 40), inhibits Ca2+ sparks and activates Ca2+ waves in the smooth muscle cells of small cerebral arteries. Our results are consistent with the idea that inhibition of Ca2+ sparks is through the activation of PKC and activation of Ca2+ waves is through other mechanisms (e.g., elevation of IP3 and [Ca2+]i). In addition, our data suggest that PKC inhibits Ca2+ sparks by decreasing the Ca2+ sensitivity of RyR channels (Fig. 9).


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Fig. 9.   Hypothetical mechanism of action of vasoconstrictors on Ca2+ signaling in arterial smooth muscle cells. Vasoconstrictors that activate phospholipase C (PLC) increase diacylglycerol (DAG), which activates protein kinase C (PKC), and increase cytoplasmic levels of inositol trisphosphate (IP3). PKC inhibits Ca2+ sparks through reducing the Ca2+ sensitivity of RyR channels and directly inhibits KCa channels. Reduction of transient KCa channel currents will lead to membrane depolarization, activation of voltage-dependent Ca2+ channels, and increased Ca2+ influx. IP3 activates IP3 receptors, which will increase Ca2+ waves. SR, sarcoplasmic reticulum; PIP2, phosphatidylinositol bisphosphate.

UTP inhibits Ca2+ sparks in cerebral artery myocytes. UTP decreased the frequency of Ca2+ sparks in the myocytes of intact cerebral arteries. It is conceivable that an increase in the frequency of Ca2+ waves could reduce the effectiveness of Ca2+ spark detection, which would lead to an observed decrease in Ca2+ spark frequency. A Ca2+ wave is a propagating wave front that occurs at a relatively low frequency in a given cell (~1 wave/10 s). The relative time that a Ca2+ wave front would occlude Ca2+ spark detection would be related to the percentage of time a Ca2+ wave front is present in a cell, and the F/F0 of the wave front (which is sufficiently high to occlude Ca2+ spark detection, see Fig. 2). In the extreme condition (i.e., +UTP), Ca2+ waves were observed in ~80% of cells during 10 s of acquisition. A Ca2+ wave front occupies approximately one-eighth of the volume of a cell and takes ~2 s to travel the distance of the cell. Therefore, Ca2+ waves alone would decrease detection of Ca2+ sparks by no more than 10%. UTP (30 µM) decreased Ca2+ spark frequency by ~85% (Fig. 1, A and B), indicating that the observed decrease in Ca2+ sparks by UTP does not occur due to occlusion of these events by Ca2+ waves. In support of this hypothesis, UTP induced a similar increase in Ca2+ wave frequency in myocytes of control arteries and in arteries that had been pretreated with the PKC inhibitor Bis I. However, in control arteries, Ca2+ spark frequency decreased with UTP, whereas, in arteries where PKC was blocked, Ca2+ spark frequency increased. These experimental findings support the idea that UTP-induced reduction of Ca2+ spark frequency is not simply due to the occlusion of these events through activation of Ca2+ waves.

There are several conceivable cellular mechanisms for UTP inhibition of Ca2+ sparks and transient KCa currents: 1) a decrease in the SR Ca2+ load that would inhibit RyR channels; 2) inhibition of voltage-dependent Ca2+ channels that would decrease cytoplasmic Ca2+ and inhibit RyR channels; 3) UTP alters the kinetics of Ca2+ sparks, such that Ca2+ release through RyR channels contributes to Ca2+ waves; and 4) UTP inhibits RyR channels and blocks Ca2+ sparks.

UTP does not reduce caffeine-induced Ca2+ transients. We have previously shown that activators of PKC do not decrease the Ca2+ load of the SR (4), but UTP could induce a decrease in SR Ca2+ load through IP3-mediated Ca2+ release. UTP did not reduce Ca2+ spark frequency through a decrease in the SR Ca2+ load, based on our observation that caffeine-induced Ca2+ transients were similar in the absence and presence of UTP. Furthermore, in the presence of the PKC blocker, UTP increased the frequency of Ca2+ sparks and transient KCa currents, and Ca2+ spark amplitude (F/F0) and transient KCa current amplitude were unaffected (Fig. 6). These effects suggest that UTP-induced IP3 elevation does not lead to a net SR Ca2+ loss.

UTP's actions are independent of voltage-dependent Ca2+ channels. UTP could conceivably decrease Ca2+ spark frequency through inhibition of L-type Ca2+ channels, which would decrease the subsarcolemmal Ca2+ concentration bathing the cytoplasmic face of RyR channels (13, 19). Indeed, the blocker of voltage-dependent Ca2+ channels, Cd2+, reduced Ca2+ spark frequency (Fig. 7). However, UTP still reduced Ca2+ spark frequency in the presence of Cd2+ to levels similar to those observed in the absence of Cd2+.

PKC mediates the effects of UTP on Ca2+ spark and transient KCa current frequency. Inhibition of Ca2+ sparks and transient KCa currents by UTP was mediated through an effect of PKC, since UTP did not inhibit Ca2+ sparks or transient KCa currents in intact arteries or isolated myocytes pretreated with Bis I. We have previously demonstrated that activators of PKC also inhibit Ca2+ sparks and transient KCa currents in isolated cerebral artery myocytes, independent of changes in SR Ca2+ load or changes in the activity of voltage-dependent Ca2+ channels (4). These results demonstrate that, although global [Ca2+]i is elevated, activation of PKC leads to a reduction in the frequency of Ca2+ sparks, which results in a mean decrease in KCa channel activity. Furthermore, our results support the idea of a direct action of PKC on RyR channels (Fig. 9; see also Ref. 41).

Evidence that UTP decreases the Ca2+ sensitivity of RyR channels. UTP decreased caffeine activation of transient KCa currents (i.e., Ca2+ sparks). Caffeine activates RyR channels through increasing the affinity of the Ca2+ activation site for Ca2+ (33) and has most commonly been applied to cells at high concentrations (>= 10 mM) to induce SR Ca2+ load depletion (e.g., see Refs. 1, 21, 34, and 49 and Fig. 5). Here we demonstrate that at a much lower concentration (25 µM) caffeine induces stable activation of transient KCa currents in cerebral artery myocytes. However, caffeine (25 µM) was unable to activate transient KCa currents when applied in the presence of UTP. A higher concentration of caffeine (500 µM), which under control conditions induced a large transient outward K+ current (probably due to rapid elevation of [Ca2+]i) (1, 21, 34), did not induce a large transient when applied in the presence of UTP but increased the frequency of transient KCa currents. Our findings therefore suggest that UTP-induced activation of PKC inhibits Ca2+ sparks through decreasing the Ca2+ sensitivity of RyR channels (Fig. 9).

UTP decreases the amplitude of transient KCa currents. We have previously reported that activators of PKC reduce transient KCa current amplitude in cerebral artery smooth muscle cells through inhibition of KCa channels (4), and PKC activators have been demonstrated to inhibit KCa channels in rat tail artery myocytes (37). Here, the inhibitory effect of UTP on transient KCa current amplitudes was prevented by pretreatment with the PKC inhibitor (Figs. 6, B and C). The reduction in transient KCa current amplitude to UTP (Fig. 4) is probably due to PKC inhibition of KCa channels, particularly since Ca2+ spark amplitude was not affected by UTP. Direct PKC inhibition of KCa channels would contribute to the mean decrease in KCa channel activity.

UTP increases the frequency of Ca2+ waves independently of PKC activation. UTP increased the frequency of Ca2+ waves in cerebral artery myocytes. UTP is known to augment Ca2+ release via a PLC-mediated (12, 39, 40) increase in the cytoplasmic concentration of IP3 (40). Furthermore, we observed a similar increase in Ca2+ wave frequency in myocytes of intact arteries when PKC was blocked with Bis I, suggesting the increase in Ca2+ wave frequency was not mediated by PKC. Ryanodine also blocked Ca2+ waves. One likely explanation for Ca2+ waves is through the combined activation of IP3 receptors by IP3 and cytoplasmic Ca2+ and of RyR channels by Ca2+. The participation of RyR channels may occur even when Ca2+ spark frequency is lowered by UTP stimulation.

cADP-ribose, a metabolite of beta -NAD, induces SR Ca2+ release through RyR channels in permeabilized porcine tracheal myocytes (35) and rabbit intestine longitudinal but not circular (24) smooth muscle. However, the physiological role of cADP-ribose-induced Ca2+ release is unclear in smooth muscle, since it does not contract permeabilized rabbit tracheal, human bronchial, or guinea pig ileum smooth muscle strips (17), but inhibition of cADP-ribose formation relaxes bovine coronary artery (8). Furthermore, cADP-ribose has been shown to inhibit IP3-mediated Ca2+ release in A7r5 cells (28) but is proposed to be necessary for initiating acetylcholine-induced [Ca2+]i oscillations in porcine tracheal smooth muscle (35). The role of cADP-ribose in vasoconstrictor-mediated Ca2+ signaling pathways remains to be established.

Physiological relevance of UTP regulation of Ca2+ sparks and Ca2+ waves. Inhibition of Ca2+ sparks with ryanodine and block of KCa channels with iberiotoxin induce a 10-mV membrane potential depolarization of cerebral arteries (23, 30). UTP depolarizes rat mesenteric artery (20) and cultured aortic smooth muscle cells (31) by 12 mV, suggesting that Ca2+ spark inhibition by UTP is a contributor to depolarization, through a decrease in the activity of KCa channels, via frequency and amplitude modulation of transient KCa currents. Depolarization will augment voltage-dependent Ca2+ channel activity, leading to increased Ca2+ influx and an increase in [Ca2+]i (Fig. 9) (23, 30). In our experiments, UTP increased the global [Ca2+]i from 119 to 179 nM (based on fractional fluorescence increase; see Ref. 19 for similar calculation), which would represent the additive effects of elevated Ca2+ influx and increased Ca2+ waves.

In summary, this study provides the first integrated view of a vasoconstrictor (UTP) action on the three Ca2+ signaling modalities (Ca2+ sparks, Ca2+ waves, global Ca2+) in smooth muscle. UTP decreased Ca2+ spark frequency and increased Ca2+ wave frequency and global Ca2+. The decrease in Ca2+ spark frequency was entirely unexpected since [Ca2+]i, an activator of RyR channels, was elevated. The inhibitory effects of UTP on Ca2+ spark frequency and associated KCa currents appeared to be mediated by a decrease of the Ca2+ sensitivity of RyR channels caused by PKC. Ca2+ waves appear to involve activation of IP3 receptors and RyR channels. Thus vasoconstrictors act through PKC and IP3 to shift Ca2+ signaling modalities from Ca2+ sparks to Ca2+ waves.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Adrian Bonev for development of the Ca2+ spark detection and analysis program.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-44455 and HL-51728 and National Science Foundation Grants IBN-9631416 and BIR-9601682, the Tutman Medical Research Fund, and a fellowship (J. H. Jaggar) from the American Heart Association, New Hampshire and Vermont Affiliate.

Address for reprint requests and other correspondence: M. T. Nelson, Dept. of Pharmacology, The Univ. of Vermont, Burlington, VT 05405 (E-mail: nelson{at}salus.med.uvm.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. Section 1734 solely to indicate this fact.

Received 21 December 1999; accepted in final form 1 May 2000.


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DISCUSSION
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Am J Physiol Cell Physiol 279(5):C1528-C1539
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