INTRODUCTION

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FORCE PRODUCTION by vascular smooth muscle cells (SMC) after activation of ₁-adrenoceptors is regulated by both Ca²⁺-dependent and Ca²⁺-independent mechanisms (9, 22). It is now being recognized that the average arterial wall Ca²⁺, observed previously (e.g., 18, 23), is not, in fact, representative of the Ca²⁺ within the individual SMC of the arterial wall, particularly during adrenergic activation. Confocal imaging of Ca²⁺ in intact arteries and veins has shown that adrenergic activation involves distinct, asynchronous intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in individual SMC (16, 19, 24, 28) rather than the abrupt initial rise and subsequent fall in average Ca²⁺ that had been recorded previously (e.g., 18). It also has been postulated (7) that Ca²⁺ sparks (25) play an important role in the ₁-adrenergic regulation of arterial diameter.

Ideally, Ca²⁺ signals of the type discussed above would be studied with high spatial resolution in blood vessels that are free to constrict and that are subjected to physiological levels of intraluminal pressure. Confocal imaging studies, however, have generally utilized arteries stretched over glass cannulas or are otherwise immobilized (i.e., are "isometric") because motion interferes with confocal imaging. Nevertheless, arteries maintained under isobaric conditions (i.e., constant pressure) and those maintained under constant length conditions (isometric) are expected to be in different physiological states. Pressurization of rat mesenteric small arteries induces myogenic tone by depolarization, with consequent entry of Ca²⁺ (20), in combination with unknown mechanisms that strongly increase the "Ca²⁺ sensitivity" of the contractile proteins (31), possibly involving protein kinase C (10). Unknown positive and negative feedback mechanisms regulating contraction during adrenergic activation are different in isometric and isobaric blood vessels (30). Therefore, in this study, we recorded Ca²⁺ signals during the motion of isobaric resistance arteries. The sole previous study of Ca²⁺ signaling in individual SMC of isobaric arteries (24) provided evidence of asynchronous Ca²⁺ transients during adrenergic activation, but did not reveal Ca²⁺ signals during active decreases in diameter or during vasomotion, which develops at high levels of agonist activation, nor were the effects on Ca²⁺ sparks observed. Neither have there been any studies of the complete time course of Ca²⁺ signals during maintained exposure to adrenergic agonists, although this is expected to be important information, given the current concept that the importance of Ca²⁺-dependent mechanisms of force declines with time (18). In the present study, therefore, we sought to record, for the first time, the Ca²⁺ signals (Ca²⁺ sparks, Ca²⁺ waves, and synchronous whole cell Ca²⁺ oscillations) within the individual SMC of isobaric arteries during decreases in diameter and during vasomotion activated by the ₁-adrenoceptor agonist phenylephrine (PE).

	METHODS

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Preparation of arteries. All experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Male Sprague-Dawley rats weighing 200-330 g were anesthetized by intramuscular injection of pentobarbital sodium (Nembutal, 50-100 mg/kg) and euthanized by cervical dislocation. The mesenteric arcade was dissected and placed in a cold dissection chamber (7°C) containing a solution (the "dissection" solution) with the following composition (mM): 2 MOPS, 145 NaCl, 4.7 KCl, 2.0 CaCl₂, 1.2 MgSO₄, 1.2 NaH₂PO₄, 0.02 EDTA, 2 pyruvate, and 5 glucose and 1.0% albumin (pH 7.4). Dissected arteries (12) were loaded with fluo 4 at room temperature for 3-4 h in the dissection solution (but without albumin) to which had been added 10 µM fluo 4-AM, 1.5% DMSO (vol/vol), and 0.03% cremophor EL (vol/vol). Arteries to be studied under isometric conditions were mounted over glass cannulas ~250 µm diameter. All arteries were equilibrated over ~1 h to initial experimental conditions (22°C, 70 mmHg) studied in a modified Krebs solution of the following composition (in mM): 112 NaCl, 25.7 NaHCO₃, 4.9 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KHPO₄, 11.5 glucose, and 10 HEPES (pH 7.4 at 22°C); equilibrated with gas of 5% O₂-5% CO₂-90% N₂. Most arteries were studied at 22°C rather than at 37°C because fluo 4 was much better retained at the lower temperature. In total, results from 35 arteries were included in this report. Initial studies in arteries free of dye at the two temperatures (see RESULTS) established some of the differences in behavior at the two temperatures.

Measurements of fluorescence and arterial diameter. We used a custom confocal laser scanning microscope described in detail previously (27, 32). The "water" objective lens (×60; numerical aperature, 1.2) provided excellent spatial resolution but a small field-of-view. To obtain a larger field-of-view during large contractions, we used a "dry" low-power objective lens (×20; 0.4 numerical aperature). Resolution with this objective lens was still adequate to resolve individual SMC. As shown in Fig. 1, the optical sections we obtained in the present study were of two types. "Radial" sections (Fig. 1A) are relatively uninfluenced by arterial wall motion and show individual SMC in cross section. A digital video (DV) clip (Fig. 1) illustrates the fact that individual SMC can be successfully "tracked" during vasomotion (DV clips for Figs. 1, 3A, and 4A are available at the AJP: Heart and Circulatory Physiology web site).¹ "Tangential" sections (Fig. 1B) (usually of arteries on glass cannulas) are strongly affected by motion but more easily reveal Ca²⁺ sparks and are required to investigate propagating Ca²⁺ waves. To improve temporal resolution when recording Ca²⁺ sparks, SMC were imaged in the tangential line-scan mode in which the same line, 50 µm in length, was scanned once every 3 ms for 1.5 s, thus creating a single line-scan image. Between successive images, however, the position of the line was moved randomly to a new position within a 50 × 50-µm planar area of the artery, a technique we refer to as "randomized confocal line scanning." This allows objective measurements of Ca²⁺ spark frequency, because the measurements cannot be biased by observer selection. With this technique, Ca²⁺ spark frequency is presented in terms of the number of Ca²⁺ sparks recorded per unit of distance (µm) scanned per unit of time (s). Measurements of arterial wall position were made either by using the edges of the fluorescence image (e.g., Fig. 1A, a and b) or in arteries not containing fluo 4, from transmitted light images recorded at 2 s¹. Fluorescence images were corrected for photobleaching of fluo 4. Although we may refer to the images as "Ca²⁺ images," all the images are simply of Ca²⁺-dependent fluo 4 fluorescence. Autofluorescence was negligible in these arteries in these experiments. All image analysis was done with custom computer procedures written in IDL (Research Systems; Boulder, CO). DV clips were constructed from the original data using digital video editing software (Adobe Systems; San Jose, CA). The DV clips may be viewed with Microsoft Windows Media Player (Microsoft; Redmond, WA).

View larger version (91K): [in this window] [in a new window]   Fig. 1.   Confocal optical sectioning of the vascular wall of pressurized mesenteric arteries during vasomotion (A) and steady vasoconstriction (B). A, left: stippled plane indicating a "horizontal radial" optical section as obtained by the confocal laser-scanning microscope. Individual smooth muscle cells (SMC) will appear in cross section in this type of image (A, a and b). This optical section is coplanar with a plane through the geometrical center of the artery. Note that the vertical component of vasomotion (arrow) is zero in this case, thus obviating movement artifacts that occur when the confocal microscope is focused in other planes. Individual SMC can be observed unambiguously during the vasomotion (A, a and b), although their shape changes. A digital video (DV) clip (DV clip is available at the AJP: Heart and Circulatory Physiology web site)1 illustrates the fact that individual SMC can be successfully "tracked" during vasomotion. B, left: stippled plane indicating a "tangential" optical section. High resolution horizontal tangential optical sections showing the individual SMC in longitudinal section (B, a). This type of optical section was used during steady vasoconstriction and in arteries mounted isometrically on glass cannulas.

	RESULTS

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Effects of temperature. Figure 2 illustrates the basic contractile responses of isobaric arteries to PE as well as some characteristics of the responses at the two different temperatures. At 37°C, exposure of isobaric arteries (70 mmHg) to relatively low concentrations of PE (300 nM) caused a decrease in diameter to a constant level, which we call "steady" vasoconstriction (Fig. 2A, top trace). Application of higher concentrations (e.g., 3.0 µM, Fig. 2A, bottom trace) caused a larger initial decrease, which was then followed by vasomotion. Cumulative concentration-effect curves are shown in Fig. 2B for arteries at 22°C (3 arteries) and 37°C (5 arteries). Oscillations were usually absent at PE concentration of <300 nM. When vasomotion was present, the average diameter was used. The two concentration-effect curves, obtained at the two different temperatures, are not significantly different (unpaired t-test, P < 0.05), both having an EC₅₀ of ~300 nM. The maximum frequency of physical oscillations (Fig. 2D) at 37°C was 0.20 ± 0.01 Hz, significantly different from that at 22°C, 0.059 ± 0.004 Hz (unpaired t-test, P < 0.05). The amplitude of the oscillations in diameter (Fig. 2C) (relative to the maximum diameter) at 37°C was also significantly larger than that at 22°C, 0.089 ± 0.003 as opposed to 0.054 ± 0.001 (unpaired t-test, P < 0.05). These results indicated that the sensitivity of the arteries to PE was not significantly different at the experimental temperature, but that the frequency and relative amplitudes of vasomotion were less at the lower temperature.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Adrenergic activation of isobaric rat mesenteric small arteries. Concentration-effect curves of phenylephrine (PE) and effects of temperature. A: time courses of vasoconstriction induced by 1-adrenoceptor agonist PE in pressurized (70 mmHg) arteries at concentrations of 300 nM (top trace) and 3.0 µM (bottom trace). The lower concentration induced a steady vasoconstriction, which relaxed rapidly upon removal of PE. At the higher concentration, the recording ended before the PE was removed. Arterial diameter refers to the outside diameter, as measured using digitized brightfield images of the arteries. B: concentration-effect curves summarizing data from the type of experiment illustrated in A, at 37°C (n = 5, open circles) and 22°C (n = 3, solid circles). Max Diameter, diameter in the absence of external Ca2+. On average, arteries developed more tone at 37°C (~16%) than at 22°C (~8%). The approximate EC50 for the two temperatures was the same, however, 300 nM. When oscillatory vasomotion occurred, the average diameter was used. C: maximum amplitude of the oscillations was much less at 22°C (~5.4% of maximum diameter) than at 37°C (~9.3% of maximum diameter). D: similarly, the maximum frequency was much less at 22°C (0.06 Hz) than at 37°C (0.20 Hz).

Ca²⁺ transients in individual SMC at low levels of adrenergically stimulated vasoconstriction. In isobaric arteries, application of PE at concentrations near the EC₅₀ caused an initial rapid, nearly synchronous Ca²⁺ transient in all the individual SMC (see Fig. 3, A and B,b, and the DV clip). At a PE concentration over the range of 300 nM-3 µM, this initial calcium transient, produced by most of the cells in an artery, varied in duration from 19 to 87 s (36.34 ± 5.05 s, n = 5 arteries). At the lower end of this range of PE concentration, the decrease in diameter then developed relatively slowly, and asynchronous Ca²⁺ transients in individual SMC then occurred, as can been seen particularly clearly in the video clip. The frequency of these oscillations and the number of cells producing such oscillations was dependent on the PE concentration. In 87 cells, taken from 4 arteries exposed to PE over the range of concentrations of 300 nM-3 µM and imaged during both the initial development of vasoconstriction and during steady vasoconstriction, the frequency of these asynchronous oscillations was 0.051 ± 0.002 Hz. It has been shown recently, in venous smooth muscle (28) and in arterial smooth muscle (33), that the number of cells producing these asynchronous Ca²⁺ transients depends on the concentration of PE. Although not quantified, it appeared that, if synchronous Ca²⁺ oscillations did not develop (see Ca²⁺ transients in individual SMC at high levels of adrenergically stimulated vasoconstriction), the frequency of the asynchronous Ca²⁺ oscillations declined throughout the period of exposure to PE.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Ca2+ and contraction recorded simultaneously during the contraction of a pressurized (70 mmHg) rat mesenteric artery stimulated with PE (300 nM). A: radial view images of the arterial wall before (top), during (middle), and after removal of PE (bottom). SMC are seen in cross section. Fluorescence intensity, indicating Ca2+-dependent fluo 4 fluorescence, is represented with the color bar ([Ca2+] increases with color from bottom to top of the color bar). Asynchronous Ca2+ transients may be seen clearly in the accompanying DV clip (DV clip is available at the AJP: Heart and Circulatory Physiology web site). B,a: position of the arterial wall, as determined from images in A, in response to stimulation. B,b: red and black traces indicate fluorescence from two identified cells that could be followed throughout the experiment. Note that fluorescence changes are synchronous initially and then become distinctly asynchronous.

Ca²+ transients in individual SMC at high levels of adrenergically stimulated vasoconstriction. Relatively high PE concentration (1.0 µM) generally resulted in vasoconstriction immediately followed by vasomotion in isobaric arteries. The spatiotemporal pattern of intracellular [Ca²⁺] was fundamentally different from that at lower PE concentrations, as illustrated in Fig. 4 and in the DV clip. Arterial diameter decreased more rapidly and to a greater extent than at lower PE concentration. The Ca²⁺ in the arterial wall was remarkably uniform during the decrease in diameter. During the oscillatory vasomotion, the peaks of the intracellular Ca²⁺ transients in different cells all coincided with each other and with the point of maximum diameter (relaxation). In seven isobaric arteries, the delay between peak Ca²⁺ and minimum diameter (maximum vasoconstriction) at concentrations of PE ranging from 300 nM to 3.0 µM was 3.0 to 19.9 s (8.17 ± 0.38 s). It has been reported that such oscillatory vasomotion requires an intact endothelium and a functional sarcoplasmic reticulum (SR) (14). To investigate the possible role of nitric oxide (NO) in these Ca²⁺ transients, we preincubated (1 h) the lumen of two arteries with inhibitors of NO synthesis (N^G-nitro-L-arginine methyl ester, 30.0 µM). No oscillatory vasomotion could be elicited by PE in these arteries.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Synchronous Ca2+ transients and oscillatory vasomotion recorded simultaneously during the contraction of a pressurized (70 mmHg) rat mesenteric artery stimulated with PE (1.0 µM). A: radial view images of the arterial wall before (top), during (middle), and after removal (bottom) of PE. SMC are seen in cross section. Synchronous nature of these Ca2+ transients may be seen clearly in the accompanying DV clip (DV clip is available at the AJP: Heart and Circulatory Physiology web site). Images are of Ca2+-dependent fluo 4 fluorescence, as represented with the color bar. B,a: position of the arterial wall, as determined from images in A. Arrowheads indicate the peak of the synchronous Ca2+ transients in B,b, below. The peak of the Ca2+ transients coincided with the maximum diameter during the oscillatory vasomotion. B,b: red, green, and black traces indicate average fluorescence from three identified cells that could be followed throughout the experiment.

Ca²+ sparks and Ca²⁺ waves during adrenergic activation. The radial sections shown in Figs. 3 and 4 do not reveal whether the asynchronous Ca²⁺ transients are actually propagating Ca²⁺ waves or what effect adrenergic activation might have on Ca²⁺ sparks. Therefore, we used arteries mounted on glass cannulas so that tangential sections could be obtained. Figure 5 illustrates the effects of exposure to high concentrations of PE on the frequency of Ca²⁺ sparks and illustrates the Ca²⁺ waves that occurred upon exposure to PE. The three images in Fig. 5A were obtained before, at 2 min during exposure to PE at a concentration of 5.0 µM, and 4 min after removal of PE. A Ca²⁺ transient was identified as a "wave" if it exhibited a constant propagation velocity for at least 20 µm. The frequency of Ca²⁺ sparks was obtained from images such as those in Fig. 5A by counting the number of Ca²⁺ sparks and dividing by the total time of scanning of the image (usually 1.5 s) and then by the total distance scanned (usually 50 µm). To be counted as a "spark," a local Ca²⁺ transient had to have a width at half-maximum of <5.0 µm and a peak amplitude (in terms of fluorescence ratio, F/F₀) of at least 1.3. Figure 5B shows the spark frequency (in each frame) as a function of time before, during, and after the exposure to PE in a typical experiment. Under the control conditions, the average frequency of Ca²⁺ sparks in six arteries during a 100-s period was 0.025 ± 0.0064 sparks · s¹ · µm¹. During the third and fourth minutes of exposure to PE (120 s), the spark frequency declined to a significantly different level, 0.0130 ± 0.0033 sparks · s¹ · µm¹ (paired t-test, P < 0.016). Histograms of the peak amplitudes (F/F₀) of the sparks are shown in Fig. 5C. No differences were found in the distribution of peak amplitudes. As a control experiment, arteries were also exposed to 30 mM KCl. In this case, the frequency of Ca²⁺ sparks increased dramatically, as expected if Ca²⁺ sparks are activated by the entry of Ca²⁺ through L-type Ca²⁺ channels (17, 21). Thus, although PE results in depolarization, a separate mechanism must exist that reduces Ca²⁺ spark frequency. During the exposure to PE, the Ca²⁺ transients were revealed to be propagating Ca²⁺ waves of the type shown in Fig. 5A, middle. The average velocity of the front of such waves in four arteries exposed to PE at a concentration of 5.0 µM was 32.4 ± 1.7 µm/s (73 waves). The waves were asynchronous between cells and did not propagate between cells.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Ca2+ sparks and Ca2+ waves recorded with randomized confocal line scanning during activation of an isometric artery by PE. A: representative random line-scan images before (a), during (b), and after (c) exposure to PE (3.0 µM). Images are of fluorescence intensity, indicating Ca2+-dependent fluo 4 fluorescence, as represented with the color bar ([Ca2+] increases with color from bottom to top). B: representative recording of the effect of PE on the frequency of spontaneous Ca2+ sparks (in sparks per second per micrometer of tissue scanned, or sparks · s1 · µm1) in a single artery. The probability of spark occurrence declined slowly after exposure to PE and recovered slowly upon its removal. C: amplitude frequency histograms for Ca2+ sparks in presence and absence of PE. There was no difference in the relative probability of finding Ca2+ sparks of a given peak amplitude under control conditions and in the presence of PE, although the absolute probability of spark occurrence was less in PE than in its absence.

	DISCUSSION

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The results support a model of adrenergic constriction of pressurized arteries in which asynchronous propagating Ca²⁺ waves underlie vasoconstriction at low levels of adrenergic activation and spatially uniform, synchronous Ca²⁺ transients underlie oscillatory vasomotion at high levels of activation. We suggest below that these two types of Ca²⁺ signals are fundamentally different in their mechanisms of origin and in their role as Ca²⁺ signals for force production. An additional component of Ca²⁺ signaling is the decline in the frequency of Ca²⁺ sparks during adrenergic activation. This may be expected to enhance vasoconstriction by further depolarizing the SMC, consequent to a reduction of Ca²⁺-activated hyperpolarizing membrane currents (25). Other possible components of Ca²⁺ signaling in arterial smooth muscle, such as the spontaneous Ca²⁺ "ripples" and "flashes" reported in wide-field imaging studies (1) were not observed in the present study, as discussed further below.

Developing and steady vasoconstriction: asynchronous, propagating Ca²+ waves. The initial release of Ca²⁺ and the asynchronous propagating Ca²⁺ waves in the isobaric arteries we studied are almost certainly dependent on ryanodine receptors and release of Ca²⁺ from the SR (2). Agonist activation of single isolated mesenteric artery myocytes with norepinephrine has shown brief Ca²⁺ transients (4, 5) dependent on SR function. Ca²⁺ waves, dependent on SR function, were elicited by norepinephrine in isolated venous myocytes (6). Propagating Ca²⁺ waves have also been recorded from isometric preparations of the rabbit inferior vena cava (28). The initial Ca²⁺ release after PE stimulation tends to occur synchronously between cells in the optical section and can account for the initial Ca²⁺ "spike" observed in previous studies that did not utilize confocal imaging techniques. The asynchronous waves that follow may then account for the lower levels of average Ca²⁺ seen in nonimaging studies (18), because asynchronous Ca²⁺ transients would not "sum" effectively. Average [Ca²⁺] would therefore be low, despite the existence of asynchronous Ca²⁺ transients. The decline in average Ca²⁺ recorded previously has been taken to indicate the development of Ca²⁺-independent mechanisms of force generation or the development of Ca²⁺ sensitization of the contractile proteins. Our experiments show that propagating Ca²⁺ waves continue to occur within individual SMC in the presence of PE concentration. This raises the possibility that Ca²⁺ is still playing a role, although the efficacy of these Ca²⁺ waves in activating force remains unknown. During the time when Ca²⁺ waves are asynchronous, the diameter may be fairly constant. Individual SMC did not appear to move independently of the arterial wall.

Vasomotion: synchronous Ca²+ oscillations. An important component of the adrenergic response is the vasomotion and the slow, spatially uniform, synchronous Ca²⁺ transients that can be seen in isobaric arteries in the presence of higher concentrations of PE. As mentioned, early studies of Ca²⁺ signals during adrenergic activation generally showed that average arterial wall [Ca²⁺]_i rose and then fell to a low maintained level, although oscillations in arterial wall [Ca²⁺] were sometimes seen (29). The present study is the first to utilize high-resolution imaging of Ca²⁺ in individual SMC during vasomotion. The synchronous Ca²⁺ oscillations we observed in the SMC were "phase-locked" to the vasomotion. The peak of Ca²⁺ elevation did not occur at the point of maximum vasoconstriction but instead during the relaxed phase of the diameter changes. The timing and synchronous nature of the Ca²⁺ elevations during vasomotion strongly suggests that the mechanism of generation involves primarily changes in membrane potential (which should be "synchronous" in different SMC). Oscillatory force production is known to be accompanied by oscillations in membrane potential (15). Depolarization could allow for coordinated L-type Ca²⁺ channel activation and Ca²⁺ influx (26). The mechanisms of oscillatory contractions or vasomotion (13) are still obscure, although it is known that vasomotion in the rat mesenteric artery requires endothelial NO release and subsequent elevation of cGMP levels in the SMC (14). Adrenergic vasoconstriction of the rat arterial mesenteric bed in situ is also associated with release of both NO and cGMP (8). It has been suggested that the stimulus for the production of NO in this case is either a rise in endothelial cell [Ca²⁺], produced by Ca²⁺ diffusing from SMC through myoendothelial gap junctions (11), or an elevation of shear stress. Shear stress was not a factor in our experiments because flow was not occurring. Regardless of the stimulus for NO production, it seems likely that NO diffuses from endothelial cells to the SMC, where it stimulates the production of cGMP and inhibits Ca²⁺ entry through L-type Ca²⁺ channels (3). The mechanism of oscillatory changes in membrane potential is not known in detail. Nevertheless, we postulate that the origin of the slow, synchronous Ca²⁺ transients involves changes in membrane potential, because only this agent would seem capable of synchronizing the activity of all the cells.

Other types of Ca²+ signals during adrenergic stimulation. Recently, two other types of spontaneous Ca²⁺ signals, "ripples" and "flashes," have been observed with the use of wide-field fluorescence microscopy, in muscle cells of the rat tail artery (1). It was suggested that the Ca²⁺ ripples are generated via inositol 1,4,5-trisphosphate-induced Ca²⁺ release in response to locally produced angiotensin II. It is possible that the frequency of ripples or proportion of SMC generating Ca²⁺ ripples and flashes could be affected by adrenergic stimulation. In the present study, however, we did not seek to determine any such possible effects. Interestingly, the amplitude of the Ca²⁺ ripples was reported to be about 0.04 (F/F₀) in peak amplitude and the Ca²⁺ oscillations induced by norepinephrine in that study were reported to have an average amplitude (F/F₀) of just <0.4. This is far less than the peak amplitude of the synchronous Ca²⁺ oscillations illustrated in Fig. 3B,b (~2.4 F/F₀). The quantitative difference in the apparent amplitude of the adrenergically stimulated Ca²⁺ transients in the two studies may reflect differences in the optical techniques used (viz. confocal vs. wide-field microscopy) or in the preparations (isobaric vs. isometric).

Effects of adrenergic stimulation on Ca²+ sparks. We observed that adrenergic activation decreased the frequency of Ca²⁺ sparks. This is in accord with the fact that activators of protein kinase C (which is activated during adrenergic stimulation) decrease Ca²⁺ spark frequency (7). It has been postulated that this is a direct effect on the ryanodine receptors, because the Ca²⁺ content of the SR (which would affect Ca²⁺ spark frequency) was not affected by activators of PKC (7). The effects of adrenergic activation on Ca²⁺ sparks within intact arteries that we have observed could be more complex, however, because Ca²⁺ content of the SR is believed to decline during adrenergic activation (4, 5) and because of possible effects other than activation of PKC, such as depolarization and increased availability of L-type Ca²⁺ channels.