RAPID COMMUNICATION
Voltage dependence of Ca2+ sparks in intact cerebral arteries

Jonathan H. Jaggar, Andrá S. Stevenson, and Mark T. Nelson

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

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Ca2+ sparks have been previously described in isolated smooth muscle cells. Here we present the first measurements of local Ca2+ transients ("Ca2+ sparks") in an intact smooth muscle preparation. Ca2+ sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). Intracellular Ca2+ concentration ([Ca2+]i) was measured in intact cerebral arteries (40-150 µm in diameter) from rats, using the fluorescent Ca2+ indicator fluo 3 and a laser scanning confocal microscope. Membrane potential depolarization by elevation of external K+ from 6 to 30 mM increased Ca2+ spark frequency (4.3-fold) and amplitude (~2-fold) as well as global arterial wall [Ca2+]i (~1.7-fold). The half time of decay (~50 ms) was not affected by membrane potential depolarization. Ryanodine (10 µM), which inhibits RyR channels and Ca2+ sparks in isolated cells, and thapsigargin (100 nM), which indirectly inhibits RyR channels by blocking the SR Ca2+-ATPase, completely inhibited Ca2+ sparks in intact cerebral arteries. Diltiazem, an inhibitor of voltage-dependent Ca2+ channels, lowered global [Ca2+]i and Ca2+ spark frequency and amplitude in intact cerebral arteries in a concentration-dependent manner. The frequency of Ca2+ sparks (<1 s-1 · cell-1), even under conditions of steady depolarization, was too low to contribute significant amounts of Ca2+ to global Ca2+ in intact arteries. These results provide direct evidence that Ca2+ sparks exist in quiescent smooth muscle cells in intact arteries and that changes of membrane potential that would simulate physiological changes modulate both Ca2+ spark frequency and amplitude in arterial smooth muscle.

ryanodine receptor; membrane potential; calcium channels

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

LOCAL CALCIUM RELEASE EVENTS through ryanodine-sensitive Ca2+ release (RyR) channels ("Ca2+ sparks") in the sarcoplasmic reticulum (SR) have been described in single smooth muscle cells enzymatically isolated from arteries (6, 26, 28), veins (1), and airway (32). Ca2+ sparks activate nearby Ca2+-sensitive K+ (KCa) channels in isolated smooth muscle cells to cause a transient outward current (26), which has previously been referred to as a "spontaneous transient outward current" (STOC) (2). STOCs cause substantial membrane potential hyperpolarizations (20 mV) in isolated smooth muscle cells (e.g., Ref. 11). It has been proposed that STOCs cause a tonic hyperpolarizing influence on the membrane potential of small pressurized arteries with myogenic tone (26), since inhibitors of Ca2+ sparks (e.g., ryanodine or thapsigargin) or of KCa channels (e.g., iberiotoxin) cause membrane potential depolarization of pressurized cerebral arteries (7, 20, 22, 26).

Intravascular pressure (e.g., from 10 to 60 mmHg) causes a graded membrane potential depolarization (from -60 to -40 mV), increases arterial wall intracellular Ca2+ concentration ([Ca2+]i; from 100 to 200 nM), and maintains constriction of small cerebral arteries (7, 20-22, 26). Inhibitors of Ca2+ sparks (e.g., ryanodine) or of KCa channels (e.g., iberiotoxin) depolarize, elevate arterial wall [Ca2+]i, and cause a tonic constriction of pressurized cerebral arteries (7, 22, 26). Ryanodine and iberiotoxin each block the effect of the other, suggesting that ryanodine receptors act through KCa channels to affect arterial membrane potential, arterial wall [Ca2+]i, and diameter (22, 26, 28). Therefore, based on isolated cell and intact artery data, it was proposed that pressure-induced membrane potential depolarization leads to an increase in the steady open probability of dihydropyridine-sensitive, voltage-dependent ("L-type") Ca2+ channels that elevates steady Ca2+ influx (26, 27, 30). The elevation of Ca2+ influx and arterial wall [Ca2+]i would lead to increased Ca2+ spark frequency and thereby STOC frequency, which would oppose the pressure-induced depolarization. Therefore, the Ca2+ spark right-arrow KCa channel pathway would be a tonic negative feedback element to regulate the membrane potential and thereby the tone of small cerebral arteries. However, Ca2+ sparks have not been identified in intact arteries, and their Ca2+ contribution to global arterial wall Ca2+ has not been directly determined in any intact smooth muscle preparation.

The frequency of STOCs in smooth muscle has been shown to increase with membrane depolarization (e.g., Refs. 2, 34). Therefore, RyR channels, and thus Ca2+ sparks, should be modulated by membrane potential through changes in the activity of voltage-dependent Ca2+ channels. Indeed, it is well established that RyR channels in muscle are activated by increases in cytoplasmic and SR Ca2+ (9, 16, 23, 24, 33, 35). Ca2+ influx evoked by depolarizing voltage steps can elicit ryanodine-sensitive changes in global [Ca2+]i in isolated smooth muscle cells, consistent with the idea that RyR channels in smooth muscle can be activated by cytoplasmic Ca2+ (e.g., Refs. 12, 13). However, the voltage dependence of Ca2+ sparks has not been determined in any smooth muscle preparation, including isolated cells.

In this study, we provide the first measurements of Ca2+ sparks in an intact smooth muscle preparation and provide the first information on the voltage dependence of Ca2+ spark frequency and amplitude in smooth muscle. Ca2+ sparks were detected in quiescent arteries, and their frequency and amplitude increased with depolarization to a membrane potential appropriate for pressurized arteries, supporting the role of such events in the negative feedback regulation of arterial diameter. This frequency and amplitude modulation of Ca2+ sparks (cf. Refs. 3, 4) by voltage depends on activation of L-type Ca2+ channels. These results indicate that Ca2+ sparks are not substantially altered by cell isolation and that their properties are modulated by voltage. This argues against the notion that Ca2+ sparks and STOCs result from single cell isolation and Ca2+ overload. The frequency and voltage dependence of Ca2+ sparks in intact arteries are consistent with Ca2+ sparks modulating smooth muscle membrane potential to regulate arterial diameter (cf. Refs. 22, 26).

    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 salt solution (PSS) 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). In all subsequent procedures, arteries were maintained in ice-cold PSS. Secondary branch posterior cerebral arteries (100-150 µm in diameter, 1-2 mm in length) were removed and cleaned of basolateral connective tissue. Arteries were carefully slipped over rectangular glass cannulas (220 µm × 40 µm × 10 mm) whose ends had been fire polished to reduce luminal arterial damage in a manner similar to that described for studies of Ca2+ oscillations in smooth muscle cells in intact tail arteries of rat (18). The measurements of Iino and colleagues (18) would not have detected Ca2+ sparks, since their image sampling rate (1 image/s) was too slow.

Ca2+ measurements. Arteries on glass cannulas were placed into a physiological K+ solution [6 mM extracellular K+ (K+o); for composition see below] containing 10 µM fluo 3-AM (Molecular Probes) and 0.05% pluronic acid (Molecular Probes) and incubated at 22°C for 60 min. After a 30-min wash at 22°C, arteries were kept on ice until required.

Arteries were imaged using a Noran Oz laser scanning confocal microscope and a ×60 water immersion lens (numerical aperture 1.2) by illumination with a krypton-argon laser at 488 nm. Images of the vessel wall (56.3 × 52.8 µm or 256 × 240 pixels) were recorded every 16.7 ms (60 images/s). For each experiment, Ca2+ spark activity was determined on the same artery before and after the application of elevated external K+ or a drug. To minimize photobleaching and cellular damage due to laser illumination, a given volume of an artery was usually scanned for only 10 s. Areas of each artery were scanned for ~20 s under control conditions, and different areas of the same artery were scanned for a total time of ~20 s in the presence of the test substance. Files were stored on writable compact disk for future analysis. To permit comparison with the single-cell data, all measurements were taken at 22°C.

Solutions. Physiological K+ solution (6 mM K+o) contained (in mM) 136 NaCl, 6 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, and 10 glucose (adjusted to pH 7.4 with NaOH). A high-K+ solution (30 mM K+o; used in this study to depolarize arteries) contained (in mM) 112 NaCl, 30 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, and 10 glucose (adjusted to pH 7.4 with NaOH). Diltiazem, thapsigargin, and wortmannin were purchased from Sigma, and ryanodine was from Calbiochem.

Analysis. Ca2+ sparks were analyzed using custom software written by Dr. Adrian Bonev in our laboratory (using IDL 5.0.2, Research Systems, Boulder, CO). To determine Ca2+ spark frequency, the frequency (Hz) of Ca2+ sparks in each artery was calculated and these means were averaged, resulting in the mean artery Ca2+ spark frequency for a given condition. Baseline fluorescence (Fo) was determined by averaging six images without activity. Local fractional fluorescence increases (F/Fo) were determined by dividing the fluorescence of an area 1.54 × 1.54 µm (7 × 7 pixels; i.e., 2.37 µm2) in each image (F) by the baseline average Fo. Ca2+ sparks were defined as local F/Fo >1.3. Such increases in fluorescence were not observed in the presence of thapsigargin or ryanodine, supporting the idea that Ca2+ sparks derive from the SR.

Estimates of Ca2+ spark and global arterial [Ca2+]i were made using the equation (8, 10)
[Ca<SUP>2+</SUP>] = <FR><NU><IT>K</IT>R</NU><DE><IT>K</IT>/[Ca<SUP>2+</SUP>]<SUB>rest</SUB> + 1 − R</DE></FR> (1)
where R is the fractional fluorescence increase (F/Fo), [Ca2+]rest is the free cytoplasmic Ca2+ concentration at Fo, and K is the apparent affinity of fluo 3 for Ca2+ (400 nM; Ref. 10). For these calculations, the reference [Ca2+]rest used in this study (119 nM) was the value determined in the same cerebral arteries under similar conditions of low pressure and PSS using the ratiometric fluorescent indicator of Ca2+ fura 2 (21, 22).

All statistical values are expressed as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Voltage dependence of Ca2+ sparks in intact arteries. Local [Ca2+]i transients (Ca2+ sparks) were observed in smooth muscle cells bathed in physiological K+ solution (6 mM K+o) and in a depolarizing solution of 30 mM K+o (Figs. 1 and 2). This elevation of external K+ increased global [Ca2+]i (fractional fluorescence increase 1.47 ± 0.12) and increased Ca2+ spark frequency 4.3-fold, from 0.24 ± 0.15 to 1.04 ± 0.28 Hz (n = 5 paired arteries; Figs. 2 and 3A). An elevation in global [Ca2+]i through membrane depolarization also causes muscle contraction (e.g., Ref. 21). Movement of the myocytes was minimized by the glass cannula. To reduce further any possible effects of muscle movement, the effects of membrane depolarization by raising external K+ from 6 to 30 mM were tested on five arteries bathed in the contractile inhibitor, 1 µM wortmannin (29). In five paired arteries bathed in 1 µM wortmannin, the frequency of Ca2+ sparks was 0.21 ± 0.11 Hz in 6 mM K+o and increased 5.5-fold to 1.15 ± 0.26 Hz with 30 mM K+o. The half time of decay (t1/2) of the Ca2+ sparks was unchanged by membrane potential depolarization (6 mM K+o, 60.7 ± 10.5 ms, n = 8 sparks; 30 mM K+o, 49.3 ± 8.3 ms, n = 41 sparks). These results indicate that local [Ca2+]i transients (Ca2+ sparks) can be measured in intact cerebral arteries and that their frequency increases with membrane depolarization.


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Fig. 1.   Ca2+ sparks in smooth muscle cells from intact rat cerebral arteries. A: 2-dimensional image of a Ca2+ spark occurring within a smooth muscle cell of an intact rat cerebral artery. Image shows fluorescence ratio (F/Fo) change that occurred over a 56.3 × 52.8-µm area of artery at the peak of a Ca2+ spark. Ratio image was obtained by dividing original confocal image by an average of 6 null images (i.e., in which a Ca2+ spark did not occur). B: 3-dimensional representation of A, illustrating changes in intracellular Ca2+ concentration ([Ca2+]i) throughout scanned area. C: mean of 40 images illustrating orientation of smooth muscle cells in artery, intensified to highlight cells with highest [Ca2+]i levels. Note that Ca2+ spark occurs in a cell with a higher resting level of [Ca2+]i.


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Fig. 2.   Depolarization increases frequency of Ca2+ sparks in intact rat cerebral arteries. A: average fluorescence over 10 s (100 images averaged) of 56.3 × 52.8-µm areas from same artery bathed in 6 mM (left) and 30 mM (right) extracellular K+ (K+o). Elevating external K+ increased global [Ca2+]i fluorescence (F/Fo) 1.62-fold in this artery. Local [Ca2+]i transients were detected by eye and are indicated by labeled boxes (1.54 × 1.54 µm). B: local F/Fo changes with time for corresponding boxes. In 6 mM K+o (left) over a 10 s period, 1 Ca2+ spark occurred (a). Changing bath solution to 30 mM K+o solution (right) increased frequency of Ca2+ sparks (10 in 10 s, a-f as indicated).


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Fig. 3.   Depolarization-induced increase in Ca2+ spark frequency in myocytes from intact cerebral arteries is reversed by Ca2+ channel blockers. A: depolarization of rat cerebral arteries by change of bath solution from 6 to 30 mM K+o increased mean frequency of Ca2+ sparks from 0.24 ± 0.15 to 1.04 ± 0.28 Hz (n = 5 paired arteries). B: diltiazem reversed depolarization-induced increase in Ca2+ spark frequency in a concentration-dependent manner. Application of 30 µM diltiazem reduced mean frequency of Ca2+ sparks compared with 5 paired control arteries (30 mM K+o) from 0.98 ± 0.11 to 0.38 ± 0.12 Hz (relative change 0.39 ± 0.11). Increasing concentration of diltiazem (60 µM) reduced Ca2+ spark frequency from 1.19 ± 0.26 to 0.18 ± 0.07 Hz (relative change 0.15 ± 0.03; 7 paired arteries).

Membrane potential depolarization also increased Ca2+ spark amplitudes. Global arterial wall [Ca2+]i has been estimated to be 119 nM in these cerebral arteries at low pressure, as measured using fura 2 (21, 22). Therefore, during a spark, local [Ca2+]i was calculated to increase from 119 to ~229 nM in arteries bathed in 6 mM K+o (Table 1), based on a fractional fluorescence change (F/Fo) of 1.59 ± 0.07 (n = 15 sparks). Membrane depolarization with 30 mM K+o increased global [Ca2+]i to ~203 nM, based on a fractional fluorescence increase of 1.47 and Fo corresponding to 119 nM. Under this depolarized condition, peak local [Ca2+]i during a Ca2+ spark was 460 nM (Table 1; F/Fo = 1.59 ± 0.02, n = 83 sparks). These results indicate that membrane depolarization increased the peak local [Ca2+]i caused by a Ca2+ spark from 229 to 460 nM (Table 1).

                              
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Table 1.   Summary of effects of membrane potential depolarization and diltiazem on steady-state [Ca2+]i, peak [Ca2+]i during a Ca2+ spark, and frequency of Ca2+ sparks

Ca2+ sparks in intact arteries are abolished by ryanodine and thapsigargin. Ca2+ sparks and STOCs in isolated cerebral artery myocytes are inhibited by ryanodine (10 µM), a blocker of the RyR channel in the SR, and by thapsigargin (100 nM), an inhibitor of the SR Ca2+ ATPase (26). Ryanodine (10 µM) and thapsigargin (100 nM) also abolished Ca2+ sparks in intact cerebral arteries. Ca2+ sparks were not observed in arteries depolarized with 30 mM K+o in the presence of ryanodine (n = 6 arteries) or thapsigargin (n = 6 arteries) (30 min exposure in 6 mM K+o). In the same arteries (in 30 mM K+o) before exposure to ryanodine or thapsigargin, Ca2+ spark frequency was 0.8 ± 0.20 or 1.14 ± 0.25 Hz, respectively.

Ryanodine and thapsigargin caused a fractional fluorescence increase of 1.23 ± 0.11 (n = 6 arteries) and 1.21 ± 0.08 (n = 6 arteries), corresponding to an elevation of global Ca2+ to 280 and 274 nM, respectively. This increase (70 nM) in global Ca2+ after application of ryanodine or thapsigargin is similar to the change in global Ca2+ (50 nM) observed with these agents on pressurized cerebral arteries, measured with fura 2 (22). The increase in global Ca2+ to ryanodine and thapsigargin appears to occur through inhibition of Ca2+ sparks, leading to membrane depolarization and subsequent Ca2+ influx through L-type voltage-gated Ca2+ channels (22, 26).

Activation of Ca2+ sparks by depolarization is reversed by Ca2+ channel blockers. Membrane potential depolarization could increase the frequency of Ca2+ sparks through activation of voltage-dependent Ca2+ channels (cf. Refs. 9, 23). This possibility was explored by examining the effects of the L-type Ca2+ channel antagonist, diltiazem, on Ca2+ sparks (diltiazem was used because it is not as light sensitive as the dihydropyridines). Ca2+ sparks were stimulated by depolarization in 30 mM K+o, and control images were acquired, after which 30 or 60 µM diltiazem was applied, and measurements were taken 3-4 min later. Diltiazem (30 µM) reduced the frequency of Ca2+ sparks in five paired arteries from 0.98 ± 0.11 to 0.38 ± 0.12 Hz, or 2.6-fold (Fig. 3B, Table 1). Diltiazem also reduced global arterial wall [Ca2+]i (F/Fo, 0.81 ± 0.05, n = 5 arteries), which corresponds to an estimated change in global [Ca2+]i from 203 to 149 nM (Table 1). Diltiazem reduced the amplitude of Ca2+ sparks (Table 1), based on a reduction in global [Ca2+]i (30 mM K+o control, 1.55 ± 0.02, n = 70 sparks; 30 µM diltiazem, 1.65 ± 0.06, n = 29 sparks). Diltiazem (30 µM) did not alter the t1/2 for Ca2+ sparks (57.6 ± 11.9 ms, n = 6 sparks).

When applied at a higher concentration, diltiazem (60 µM) reduced Ca2+ spark frequency 6.6-fold [control (30 mM K+o), 1.19 ± 0.26 Hz; 60 µM diltiazem, 0.18 ± 0.07 Hz; 7 paired arteries; Fig. 3B], and reduced global arterial wall [Ca2+]i (F/Fo, 0.48 ± 0.01) from 203 to 77 nM (Table 1). The mean peak amplitude of local [Ca2+]i during a Ca2+ spark was also reduced by 60 µM diltiazem from 480 nM in control (30 mM K+o; F/Fo 1.62 ± 0.02, n = 157 sparks) to 173 nM in 60 µM diltiazem (F/Fo 1.87 ± 0.07, n = 25 sparks) (Table 1). Therefore, the change in local [Ca2+]i due to a Ca2+ spark was reduced from 277 nM in 30 mM K+o to 96 nM by 60 µM diltiazem. The t1/2 for Ca2+ sparks that occurred in the presence of 60 µM diltiazem was unchanged (64.3 ± 17.1 ms, n = 12 sparks). These results suggest that activation of voltage-dependent Ca2+ channels is involved in the increase in Ca2+ spark frequency and amplitude caused by membrane potential depolarization.

Ca2+ sparks in cerebral arterioles. To determine whether Ca2+ sparks occurred in smaller diameter arteries, a 40- to 50-µm side branch (~250 µm in length) attached to a secondary branch posterior cerebral artery was oriented under the main vessel so that the whole length of the branch could be imaged. Ca2+ sparks were also resolved in this smaller diameter arteriole (Fig. 4). With 30 mM K+o, diltiazem (30 µM) reduced Ca2+ spark frequency from 2.03 ± 0.54 to 0.43 ± 0.17 Hz. The mean peak F/Fo of the Ca2+ sparks was relatively unchanged, with F/Fo of 1.74 ± 0.04 in control (n = 81 sparks) and 1.70 ± 0.06 in the presence of 30 µM diltiazem (n = 17 sparks). These results suggest that Ca2+ sparks also exist in cerebral arterioles.


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Fig. 4.   A: Ca2+ sparks in an intact cerebral arteriole in a bathing solution containing 30 mM K+o. B: corresponding changes in F/Fo with time.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

This study presents the first recordings of Ca2+ sparks from an intact smooth muscle preparation, namely resistance-sized (40-150 µm in diameter) cerebral arteries. Ca2+ sparks appeared to be caused by the opening of RyR channels in the SR, based on inhibition by ryanodine and thapsigargin and on their subcellular localization (10, 26). Membrane potential depolarization of intact arteries with elevated K+ (30 mM) increased Ca2+ spark frequency and amplitude, and this effect was reduced in a concentration-dependent manner by the Ca2+ channel inhibitor diltiazem. These results indicate that Ca2+ sparks are regulated by Ca2+ entry through voltage-dependent Ca2+ channels, as has been observed in cardiac muscle (9, 23).

Ca2+ sparks in intact arteries and isolated cells. A major advance of the technique described in this study is the ability to examine local [Ca2+]i signals with millisecond time resolution in intact arteries. Enzymatic separation of single smooth muscle cells from intact tissue leads to loss of intercellular communication, as well as possible cellular damage. Our approach permits the simultaneous measurement of Ca2+ sparks in a number (~8) of smooth muscle cells and thereby increases the amount of data that can be collected during scanning. Ca2+ spark properties (frequency, F/Fo, t1/2) appear to be similar in the intact arteries and in cells enzymatically isolated from the same arteries (6, 26, 28). Ca2+ spark frequency in intact arteries ranged, depending on voltage, from ~0.2 to 1.2 Hz in a volume that corresponds to about two to four cells, or 0.05-0.6 Hz/cell. Ca2+ spark frequency was ~1 Hz in isolated cerebral artery myocytes (6, 28) when the scanned volume was taken into account. STOC frequency in cerebral artery myocytes was also ~1 Hz when the cells were voltage clamped at -40 mV, a voltage similar to the membrane potential of isolated myocytes and smooth muscle cells in pressurized (60 mmHg) arteries (6, 7, 21, 26, 28). Therefore, Ca2+ spark frequency in intact arteries depolarized to about -40 mV (30 mM K+o) was in the range of that measured in isolated myocytes at similar voltages (~0.2-1.0 Hz/cell), particularly in light of uncertainties of precise voltages, [Ca2+]i, and scan volumes. Fractional fluorescence changes during a Ca2+ spark and t1/2 were similar in intact arteries and isolated myocytes (F/Fo 1.5-1.9, t1/2 ~50 ms) (6, 28).

Ca2+ sparks were recorded in quiescent (noncontracted) arteries (Figs. 2 and 3) bathed in PSS, arguing against the notion that Ca2+ sparks appear only under nonphysiological conditions of [Ca2+]i overload of isolated cells. Therefore, our results demonstrate that Ca2+ sparks occur in intact cerebral arteries.

Membrane depolarization increases Ca2+ spark frequency and amplitude in intact arteries. In the present study, the effects on membrane potential of elevating pressure to ~60 mmHg were simulated by depolarizing with 30 mM K+. Elevation of external K+ from 6 to 30 mM depolarizes smooth muscle cells in arteries from approximately -60 mV to -40 mV (Refs. 5, 14; for review see Ref. 17), similar to the depolarizing effect of increasing intravascular pressure to ~60-80 mmHg (7, 15, 20, 21, 26). Based on the fractional fluorescence change and an arterial wall [Ca2+]i of 119 nM in 6 mM K+ (21), this increase in external K+ raised global arterial [Ca2+]i to 203 nM, a value similar to what was measured (190 nM) with fura 2 in the same arteries subjected to 60 mmHg (21). Elevated K+ also increased Ca2+ spark frequency (4.3-fold) and amplitude (from an estimated peak [Ca2+]i of 229-460 nM). This membrane potential depolarization-induced increase in Ca2+ spark frequency and amplitude, as well as the increase in global [Ca2+]i, should have a profound effect on the activity of KCa channels, which are presumably responsible for the observed iberiotoxin- and ryanodine-sensitive change in membrane potential measured in intact pressurized arteries. These results are consistent with the idea that Ca2+ sparks and KCa channels form a negative feedback element to limit tonic membrane depolarization and constriction of cerebral arteries (cf. Ref. 26).

Membrane depolarization from -60 to -40 mV has been shown to increase Ca2+ spark frequency in cardiac myocytes (9, 31). This increase in Ca2+ spark frequency appears to be largely due to activation of Ca2+ sparks by local Ca2+ entry through dihydropyridine-sensitive, voltage-dependent Ca2+ channels (9, 23, 31). RyR channel open probability in muscle SR appears to be regulated by cytoplasmic Ca2+ (33), and this Ca2+ sensitivity may be further modulated by SR [Ca2+] (16, 24, 35, 33). Steady membrane depolarization of arterial smooth muscle increases voltage-dependent Ca2+ channel open probability (e.g., 30), global [Ca2+]i, and presumably SR [Ca2+]. Therefore, an elevation in local [Ca2+]i from Ca2+ channels, global [Ca2+]i, or SR [Ca2+] could contribute to the observed depolarization-induced increase in Ca2+ spark frequency. The contributions of these different mechanisms to the increase in Ca2+ spark frequency remain to be determined.

Direct contribution of Ca2+ sparks to global [Ca2+]i in intact cerebral arteries. Myogenic cerebral arteries subjected to physiological levels of intravascular pressure (e.g., 60 mmHg) have steady membrane potentials of about -40 mV, arterial wall [Ca2+]i of ~200 nM, and are constricted by ~30% (7, 20, 21, 25, 26). Under conditions designed to simulate the effects of pressure on membrane potential and arterial wall [Ca2+]i (i.e., 30 mM K+o), Ca2+ spark frequency was ~1 s-1 in a volume that approximately corresponds to two to four cells. One Ca2+ spark would raise global [Ca2+]i in a single cell by <2 nM (see Ref. 26). Ca2+ release through RyR channels can contribute significantly to global [Ca2+]i in cerebral and coronary artery myocytes, if it is evoked by nonphysiological depolarizing voltage steps with elevated levels of external Ca2+ (>2 mM) (13, 19) or by bolus addition of caffeine (13, 19, 22). Nonetheless, under the conditions used in our experiments (i.e., at room temperature), direct Ca2+ spark measurements under steady-state conditions appropriate for pressurized myogenic cerebral arteries indicate that RyR channels do not contribute sufficient Ca2+ spatially and temporally to directly alter global [Ca2+]i. Furthermore, in the presence of a KCa channel blocker (iberiotoxin), ryanodine was without effect on arterial diameter and wall [Ca2+]i, also arguing against a significant direct contribution of RyR channels to steady-state global [Ca2+]i (22). However, it is conceivable that under other conditions (e.g., in the presence of vasoconstrictors) Ca2+ sparks, perhaps in the form of Ca2+ waves, could contribute significantly to global Ca2+ changes in intact arteries (1).

In summary, we show that Ca2+ sparks occur in intact cerebral arteries at physiological membrane potentials and exhibit similar frequencies, amplitudes, and t1/2 of those previously described in isolated cerebral artery smooth muscle cells (6, 26, 28). These results provide evidence that the steady-state frequency and amplitude of Ca2+ sparks is modulated through the activity of voltage-dependent Ca2+ channels in intact cerebral arteries. Furthermore, this study supports the idea that membrane potential depolarization leads to an elevation of Ca2+ spark frequency and amplitude, which increases STOC (KCa channel) frequency and amplitude, thereby applying a membrane potential hyperpolarizing "brake" on vasoconstriction. The ability to examine Ca2+ sparks in intact arteries should provide considerable new insights into vascular function.

    ACKNOWLEDGEMENTS

We thank Dr. Adrian Bonev, Dr. Joseph Brayden, and Dr. George Wellman for comments on the manuscript.

    FOOTNOTES

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

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.

Address for reprint requests: M. T. Nelson, Dept. of Pharmacology, The University of Vermont, Burlington, VT 05405.

Received 20 February 1998; accepted in final form 19 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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