December 2004
Volume 45, Issue 12
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Physiology and Pharmacology  |   December 2004
Identification and Spatiotemporal Characterization of Spontaneous Ca2+ Sparks and Global Ca2+ Oscillations in Retinal Arteriolar Smooth Muscle Cells
Author Affiliations
  • Tim M. Curtis
    From the Ophthalmic Research Centre, The Queen’s University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Belfast, Northern Ireland; and the
  • James Tumelty
    From the Ophthalmic Research Centre, The Queen’s University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Belfast, Northern Ireland; and the
    Smooth Muscle Group, The Queen’s University of Belfast, Medical Biology Centre, Belfast, Northern Ireland.
  • Jennine Dawicki
    From the Ophthalmic Research Centre, The Queen’s University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Belfast, Northern Ireland; and the
  • C. Norman Scholfield
    Smooth Muscle Group, The Queen’s University of Belfast, Medical Biology Centre, Belfast, Northern Ireland.
  • J. Graham McGeown
    Smooth Muscle Group, The Queen’s University of Belfast, Medical Biology Centre, Belfast, Northern Ireland.
Investigative Ophthalmology & Visual Science December 2004, Vol.45, 4409-4414. doi:10.1167/iovs.04-0719
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      Tim M. Curtis, James Tumelty, Jennine Dawicki, C. Norman Scholfield, J. Graham McGeown; Identification and Spatiotemporal Characterization of Spontaneous Ca2+ Sparks and Global Ca2+ Oscillations in Retinal Arteriolar Smooth Muscle Cells. Invest. Ophthalmol. Vis. Sci. 2004;45(12):4409-4414. doi: 10.1167/iovs.04-0719.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To identify spontaneous Ca2+ sparks and global Ca2+ oscillations in microvascular smooth muscle (MVSM) cells within intact retinal arterioles and to characterize their spatiotemporal properties and physiological functions.

methods. Retinal arterioles were mechanically dispersed from freshly isolated rat retinas and loaded with Fluo-4, a Ca2+-sensitive dye. Changes in [Ca2+]i were imaged in MVSM cells in situ by confocal scanning laser microscopy in x-y mode or line-scan mode.

results. The x-y scans revealed discretely localized, spontaneous Ca2+ events resembling Ca2+ sparks and more global and prolonged Ca2+ transients, which sometimes led to cell contraction. In line scans, Ca2+ sparks were similar to those previously described in other types of smooth muscle, with an amplitude (ΔF/F0) of 0.81 ± 0.04 (mean ± SE), full duration at half maximum (FDHM) of 23.62 ± 1.15 ms, full width at half maximum (FWHM) of 1.25 ± 0.05 μm, and frequency of 0.56 ± 0.06 seconds−1. Approximately 35% of sparks had a prolonged tail (>80 ms), similar to the Ca2+“embers” described in skeletal muscle. Sparks often summated to generate global and prolonged Ca2+ elevations on which Ca2+ sparks were superimposed. These sparks occurred more frequently (2.86 ± 025 seconds−1) and spread farther across the cell (FWHM = 1.67 ± 0.08 μm), but were smaller (ΔF/F0 = 0.69 ± 0.04).

conclusions. Retinal arterioles generate Ca2+ sparks with characteristics that vary during different phases of the spontaneous Ca2+-signaling cycle. Sparks summate to produce sustained Ca2+ transients associated with contraction and thus may play an important excitatory role in initiating vessel constriction. This deserves further study, not least because Ca2+ sparks appear to inhibit contraction in many other smooth muscle cells.

As with all vascular systems, blood flow through the retinal circulation depends on the perfusion pressure gradient across the vascular bed and the resistance to flow within it. Vascular resistance is inversely related to the fourth power of the radius of a blood vessel. Thus, a small change in diameter has a substantial influence on the blood flow. Variation in vessel diameter occurs frequently and may be considered the main regulatory mechanism controlling flow in the retinal circulation. 1 The retinal arterioles provide the major site of resistance to blood flow in the retina and thus have the greatest capacity for regulation of retinal perfusion. 2 Alterations in retinal arteriolar tone occur through the contraction or relaxation of the microvascular smooth muscle (MVSM) cells in the wall of the vessels. The retinal vasculature is not innervated, 3 and retinal arteriolar tone is mainly regulated by local factors, such as variations in pO2, pCO2, and pH, as well as mediators released from neighboring endothelial and retinal cells (e.g., nitric oxide and endothelin-1). 1 These local influences combine to ensure that blood flow to the retinal tissue is closely matched to metabolic demand. 
Although some of the local factors responsible for regulating retinal vessel diameter have been identified, as just listed, the complex cellular mechanisms that underlie changes in retinal MVSM tone are poorly understood. MVSM contractility is known to be heavily dependent on changes in intracellular calcium ([Ca2+]i). 4 5 We have shown that retinal MVSM cells possess a variety of Ca2+-handling mechanisms that modulate cytosolic [Ca2+], including the voltage-dependent and store-operated Ca2+ influx channels, 6 pools of releasable Ca2+ (the sarcoplasmic reticulum [SR] Ca2+ stores), 7 and multiple Ca2+ efflux pathways. 8  
Our previous studies were largely restricted to the measurement of average retinal MVSM [Ca2+]i. The application of confocal Ca2+-imaging techniques have demonstrated, however, that [Ca2+]i signals are not homogenously distributed, and increases in [Ca2+]i can differ in relation to their spatiotemporal properties and physiological functions. Both local and global Ca2+-signaling events have been observed in vascular smooth muscle cells, and these may be generated spontaneously 9 or in response to agonist exposure. 10 11 Localized Ca2+ transients are thought to result from the opening of clusters of ryanodine-sensitive Ca2+ release channels on the SR and have been termed Ca2+ sparks. By activating Ca2+-activated K+ channels (KCa) to generate spontaneous outward currents (STOCs), Ca2+ sparks are thought to inhibit smooth muscle contraction in many tissues by increasing membrane polarization and deactivating voltage-dependent Ca2+ channels, thus reducing Ca2+ influx. 12 In contrast to Ca2+ sparks, global Ca2+ oscillations have been implicated as mediators of constriction. 13 The frequency of these Ca2+ oscillations has been shown to increase with agonists that elevate inositol 1,4,5 triphosphate (IP3) levels, suggesting that Ca2+ release through IP3 receptors on the SR contributes, at least in part, to their generation. 10 13  
The goal of the present study was to determine the spatiotemporal characteristics and functional relevance of spontaneous Ca2+-transients in retinal MVSM by obtaining the first images from retinal arterioles of both localized and generalized Ca2+ signaling events. Furthermore, we show that Ca2+ sparks can summate to produce sustained global Ca2+ oscillations, some of which are followed by cell contraction. This is consistent with a model in which local Ca2+ release events play an excitatory, rather than an inhibitory, role in the retinal vasculature, and thus runs counter to current paradigms concerning the functional significance of Ca2+ sparks in vascular smooth muscle. 12 We also demonstrate the existence of two distinct populations of Ca2+ sparks for the same release sites during different phases of spontaneous signaling, presumably reflecting changes in localized Ca2+-release with different levels of cytoplasmic- and SR-[Ca2+]. 
Methods
Retinal Microvessel Preparation
Male Sprague-Dawley rats (200–300 g) were anesthetized with CO2 and killed by cervical dislocation. Animal use conformed to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and UK Home Office Regulations. Retinas were rapidly removed and arterioles devoid of surrounding neuropile isolated as previously described. 7 6 In brief, retinal quadrants were lightly triturated using a fire-polished Pasteur pipette (internal tip diameter, 0.3 mm) in a low-Ca2+ Hanks’ solution. Homogenates were centrifuged at 3000 rpm for 1 minute, the supernatant aspirated off, and the tissue washed again with low-Ca2+ medium. The remaining fragments were incubated at 21°C in 1 mL of low-Ca2+ Hanks’ solution containing 10 μM of Fluo-4 AM, a Ca2+-sensitive dye (Molecular Probes, Eugene, OR) and the suspension agitated every 15 minutes for 2 hours. This prolonged incubation was necessary to facilitate adequate loading of the retinal MVSM cells with Fluo-4. 
Homogenates were diluted with 10 volumes of low-Ca2+ medium and the mixture vigorously triturated. Of this mixture, 1 mL was pipetted into a rotatable circular glass-bottomed recording bath on the stage of an inverted microscope (Eclipse TE300; Nikon, Tokyo, Japan). Microvessels were anchored with tungsten wire (50 μm diameter, 2 mm length) and superfused with normal Hanks’ solution at 37°C. The recording bath was rotated so that the long axis of the arterioles was parallel to the x-axis of the microscope. Drug solutions were delivered via a five-way micromanifold with an exchange time of ∼1 second, as measured by switching to a dye solution. 
Solutions
The bath solution had the following composition (in mM): 140, NaCl; 5, KCl; 5, d-glucose; 2, CaCl2; 1.3, MgCl2; 10, HEPES (pH 7.4) with NaOH. Low-Ca2+ medium differed only in that it contained 0.1 mM CaCl2
Ca2+ Imaging and Data Analysis
Changes in [Ca2+]i were imaged in MVSM cell arrays with a confocal scanning laser microscope (MR-A1; Bio-Rad, Richmond, CA) used in x-y mode at a rate of 1 image per 1.2 seconds and in line scan mode at a rate of 500 scans per second. 14 Confocally imaged microvessels were excited at 488 nm, and emitted light was filtered through a 530- to 560-nm band-pass filter. Data acquisition was controlled by computer (Timecourse software; Lasersharp; Bio-Rad) and images were processed and analyzed on computer (Image J; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-imageJ; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Confocal fluorescence data (F) were normalized to the average resting fluorescence (F0) for periods that exhibited no spontaneous elevations in [Ca2+]i
Ca2+ sparks were measured within regions of interest (ROIs; 4 × 4-μm boxes in x-y mode and 4-μm ×120-ms boxes in line-scan mode), while global Ca2+ oscillations were averaged across the entire cell. Ca2+ events were defined as an increase in F/F0 of >2 standard deviations above the mean resting fluorescence. The amplitudes of Ca2+ sparks and global Ca2+ oscillations were taken as the maximum increase in normalized fluorescence (ΔF/F0). Ca2+ event durations were measured along a line through the peak fluorescence as the time elapsed from reaching half the maximum amplitude during the rising phase, to falling back to that value during the decay (i.e., the full duration at half-maximum fluorescence (FDHM). Spatial spread was similarly defined as the distance in micrometers between the half-maximum fluorescence increase on either side of the peak fluorescence (i.e., the full width at half-maximum [FWHM]). In some experiments using x-y scan mode, cell area was calculated by using the area calculator plug-in in Image J. 
Data are expressed as the mean ± SE. Comparisons were made with unpaired Student’s t-tests, with P < 0.05 considered significant. 
Results
Identification of Ca2+ Sparks and Global Ca2+ Oscillations in Retinal MVSM Cells
To explore whether distinct subcellular Ca2+ transients exist in rat retinal MVSM cells, we monitored changes in fluorescence intensity by laser scanning confocal microscopy in myocytes still embedded within their parent arterioles and loaded with the Ca2+ indicator dye Fluo-4 (Molecular Probes). For the purposes of this study, recordings were confined to retinal arteriole segments that were 35 to 40 μm in diameter, which represent the main trunk arterioles that emanate from the optic disc. 6 The wall of isolated retinal arterioles consisted of a monolayer of MVSM cells surrounding an intact endothelium (Fig. 1) . Under resting conditions, vascular myocytes within freshly dispersed retinal arterioles demonstrated considerable Ca2+-signaling activity. Two main types of spontaneous [Ca2+]i transients were observed, with different kinetics and spread. Brief and spatially localized events resembling Ca2+ sparks were often seen in close proximity to the cell membrane, as well as more prolonged global Ca2+ oscillations which usually spread across the full width of the cell (Fig. 2)
Global Ca2+ Oscillations and Retinal MVSM Contraction
In arterial smooth muscle, global Ca2+ oscillations have been recognized as the main driving force underlying vasoconstriction. 13 Likewise, we observed that spontaneous global Ca2+ oscillations in retinal MVSM cells triggered contractile responses. In the example shown (Fig. 3) , a global Ca2+ oscillation was associated with a 19% reduction in MVSM cell area. Although it is apparent, therefore, that a prolonged global increase in [Ca2+]i can trigger retinal MVSM contraction, this was relatively uncommon. In six vessels (82 cells), only 18 of 103 global Ca2+ oscillations were followed by a decrease in cell area. The amplitude of the [Ca2+]i increases was no higher in cells that contracted than in those that did not (P = 0.55), and so this variability probably reflects differences in the sensitivity of the individual cells to [Ca2+]i. Mechanical contraction was never observed in response to individual Ca2+ sparks (6 vessels, 82 cells, 163 sparks). 
Ca2+ Sparks and Global Ca2+ Oscillations
Although spontaneous [Ca2+]i transients were visualized in retinal MVSM cells by using x-y scan imaging, this approach did not allow adequate temporal resolution of rapid, localized [Ca2+]i changes within a cell. Consequently, it appears from Figure 2B that both Ca2+ sparks and global Ca2+ oscillations have a similar time course. To improve temporal resolution, we used the line-scan mode of confocal imaging. Analysis was limited to vessel segments exhibiting no frame-to-frame movement of the cells. A typical example of an image obtained by scanning a line oriented at right angles to the long axis of a cell is shown in Figure 4A . A brief, localized Ca2+ spark can be clearly seen in the image. This spontaneously localized Ca2+ event had a peak amplitude (ΔF/F0) of 0.95 and an FDHM of 14 ms and was restricted to a relatively small area (FWHM of 1.57 μm). Some Ca2+ sparks (≅35% of recorded sparks in seven cells) were associated with a prolonged tail (>80 ms) similar to that in the example in Figure 4A . These events resemble the Ca2+“embers” or “glows” recently described in skeletal muscle cells. 15 Ca2+ embers were site dependent and, in some sites, practically all Ca2+ sparks displayed prolonged tails. 
It has been demonstrated in ileal myocytes that spatiotemporal recruitment of “elementary” Ca2+ sparks may give rise to cell-wide elevations in [Ca2+]i. 16 Using line-scan imaging, we were able to visualize the initiation site of some global Ca2+ oscillations in retinal MVSM cells. In the example shown in Figure 4B it is evident that a global Ca2+ oscillation was initiated from a site where spontaneous Ca2+ sparks were also observed. The temporal profile of the global Ca2+ oscillation consisted of a series of step-like increases in fluorescence resulting from the summation of consecutive Ca2+ sparks. It is also striking that even after the global Ca2+ oscillation reached its maximum, Ca2+ sparks persisted throughout both the plateau and declining phases. 
Two Populations of Ca2+ Sparks
The spatiotemporal properties of Ca2+ sparks and global Ca2+ oscillations in retinal MVSM cells were characterized from line-scan images of 60 cells in 6 vessels, and the data are summarized in Table 1 . Ca2+ sparks were separated into two groups: “basal” sparks that arose from resting fluorescence values (F/F0 of 0.95–1.05) and those that were superimposed on global Ca2+ oscillations (Ca2+ sparks on oscillations). At 0.81, the mean spark amplitude under basal conditions was nearly six times the SD of the background signal noise (noise SD = 0.138). Global Ca2+ oscillations were similar in amplitude to basal Ca2+ sparks, but were nearly 100-times longer in duration (as measured from the FDHM) and occurred less frequently (Table 1) . Differences in the spatiotemporal properties of basal Ca2+ sparks and Ca2+ sparks overlaying Ca2+ oscillations were also observed. The latter were smaller in amplitude, increased in width, and increased in frequency (Table 1)
Discussion
In this study, we describe the first visualization of spontaneous subcellular Ca2+ transients in retinal arteriolar smooth muscle cells. Two distinct Ca2+ signaling events were seen, discretely localized: spontaneous near-membrane Ca2+ sparks and more global and prolonged Ca2+ oscillations. In most studies on elementary Ca2+-signaling events in arterial smooth muscle, investigators have used single, isolated myocytes, even though the ultimate goal is to understand how function is regulated in intact vessels. 10 17 A major advantage of the technique described herein is the use of intact arteriole segments in which the physiological relationships between the retinal MVSM cells, basal lamina, and endothelium are preserved. Our technique also allowed simultaneous imaging of subcellular Ca2+ signals in several MVSM cells, thus allowing cell-to-cell variation to be assessed while increasing the amount of data that can be collected in a single experiment. 
Ca2+ sparks are thought to result from transient local release of Ca2+ from intracellular stores and have been described in cardiac, 18 skeletal, 19 and several smooth muscle cell preparations, 12 including arteriolar smooth muscle cells. 20 In smooth muscle cells, Ca2+ spark amplitudes are known to be quite variable, with the average peak increase in [Ca2+]i ranging from 50 to 200 nM. 12 In retinal MVSM cells, the average spark amplitude (ΔF/F0 of 0.81; Table 1 ) equates to an elevation in [Ca2+]i of ∼80 nM, as determined using the pseudoratiometric calculation of Cheng et al., 18 assuming an in situ dissociation constant for Fluo-4 of 1000 nM 21 and a resting Ca2+ level in retinal arterioles of 66 nM. 7 The average frequency (0.56 second−1), duration (23.6 ms FDHM), and spatial spread (1.25 μm FWHM) of Ca2+ sparks in retinal MVSM are all similar to those in other smooth muscle cells, with reported values for these parameters ranging from 0.5 to 1 second−1, 12 30 to 65 ms, 22 and 1.2 to 2.3 μm, 23 24 respectively. There are, however, some reports of more prolonged Ca2+ sparks (100–600 ms) in tracheal 25 and urinary bladder smooth muscle cells, 26 whereas the events observed in human cerebral arterial smooth muscle cells appeared to spread further, with an average FWHM of 8.2 μm. 27  
Our records provide clear evidence that Ca2+ sparks in retinal MVSM cells fuse to produce cell-wide global Ca2+ oscillations that can lead to cell contraction. These findings are of particular interest, because they imply that Ca2+ sparks in retinal arterioles are principally excitatory in nature, whereas it has been proposed that Ca2+ sparks exert a predominantly inhibitory effect in vascular smooth muscle, providing a negative feedback mechanism that favors decreased Ca2+ influx and vasodilatation. 12 Only a small proportion of global Ca2+ oscillations actually led to retinal MVSM cell contraction. Ca2+ ions regulate nearly every cell function, and subcellular Ca2+ transients are known to cause a pulsatile activation of Ca2+-dependent enzymes, 28 and to drive changes in gene expression. 29 Consequently, those Ca2+ oscillations that failed to initiate excitation–contraction coupling are still likely to be physiologically relevant. 
Detailed analysis revealed two distinct populations of Ca2+ sparks, with those superimposed on global Ca2+ oscillations displaying an increased frequency and spread, but reduced amplitude, when compared with sparks originating at the same release sites but from basal [Ca2+]i levels. Ca2+ sparks are thought to be generated by the opening of ryanodine receptor-linked channels (RyRs) on the SR, 12 and elevations in cytosolic Ca2+ are known to increase RyR open probability. 30 This may well explain the increased spark frequency during global Ca2+ oscillations. Likewise, increased spatial spread may also be accounted for by an overall increase in the open probability of RyRs, since this would favor recruitment of release sites. Because global Ca2+ oscillations in smooth muscle cells are known to involve Ca2+ store release, 13 the smaller amplitudes of the Ca2+ sparks during such oscillations in retinal MVSM cells may reflect a reduction in SR Ca2+ content. Clearly, further studies are now needed to unravel the precise mechanisms through which localized Ca2+ release is modified during Ca2+ oscillations, as well as to determine the functional implications of such modifications. 
Under basal conditions, many sparks in retinal MVSM cells had protracted tails similar to the Ca2+ embers of skeletal muscle cells. 15 We have described prolonged, spontaneous Ca2+ release events in isolated smooth muscle cells during store-overload, 14 but no such events have been reported in any intact tissue. In skeletal muscle cells, embers are thought to reflect direct RyR opening by voltage sensors 15 but, because the RyRs are not believed to be under direct voltage control in smooth muscle, it is unclear what mechanism generates Ca2+ embers in retinal MVSM cells. It seems likely, however, that events in which Ca2+ release is prolonged well beyond the average channel-open time associated with RyRs in lipid bilayer experiments 31 may have important consequences, perhaps increasing the likelihood of spark summation and the initiation of global Ca2+ oscillations. These findings also underline the fact that the study of intact tissues may reveal subtleties of signaling behavior not apparent in isolated cells or molecules. 
A possible limitation of our current model is the use of nonpressurized retinal arterioles. Passive stretching of the retinal vessel wall during increases in intraluminal pressure may lead to an elongation of the MVSM cells and thereby provoke changes in the spatiotemporal properties of the spontaneous Ca2+ signals. Increases in retinal MVSM cell length could modulate spontaneous Ca2+ sparks and global oscillations through the activation of stretch-activated currents 32 or via stretch-induced gating of RyRs. 33 In rat cerebral arteries, pressurization increases the frequency of Ca2+ sparks and global Ca2+ oscillations, but other spatiotemporal features such as amplitudes and rise times are similar to those in nonpressurized vessels. 34  
In summary, the application of high-resolution imaging to intact retinal arterioles has allowed us to visualize subcellular Ca2+-signaling events in retinal MVSM cells. These cells are the primary effectors of retinal arteriolar tone, and the data from the present study takes us a step closer to elucidating the basic mechanisms involved in the regulation of local blood flow in the retina. Understanding how such control is achieved will be fundamental to the development of novel therapeutic strategies designed to restore adequate blood flow in disease states, such as diabetic retinopathy 35 and glaucoma. 36  
 
Figure 1.
 
Luminal confocal section of an isolated retinal arteriole loaded for 10 minutes with the membrane-tracking dye, di-4-ANEPPS (Molecular Probes, Eugene, OR). Image dimensions = 84 μm × 98 μm.
Figure 1.
 
Luminal confocal section of an isolated retinal arteriole loaded for 10 minutes with the membrane-tracking dye, di-4-ANEPPS (Molecular Probes, Eugene, OR). Image dimensions = 84 μm × 98 μm.
Figure 2.
 
Ca2+ sparks and waves imaged in Fluo-4–loaded retinal MVSM cells. (A) Four time frames from a series of x-y images of a retinal arteriole. The MVSM cells are oriented at right angles to the long axis of the vessel. Fluorescence relative to basal values (F/F0) is represented in grayscale, as indicated on the calibration bar on the left. Four ROIs are marked, and the average fluorescence within each of these regions through the course of the 40-second experiment is plotted against time in (B). Arrows: the time point at which each frame was captured. Both small localized events (ROIs 3, 4), and generalized Ca2+ oscillations (ROIs 1, 2) were seen. Movie 1 shows the spontaneous Ca2+ increases in this vessel.
Figure 2.
 
Ca2+ sparks and waves imaged in Fluo-4–loaded retinal MVSM cells. (A) Four time frames from a series of x-y images of a retinal arteriole. The MVSM cells are oriented at right angles to the long axis of the vessel. Fluorescence relative to basal values (F/F0) is represented in grayscale, as indicated on the calibration bar on the left. Four ROIs are marked, and the average fluorescence within each of these regions through the course of the 40-second experiment is plotted against time in (B). Arrows: the time point at which each frame was captured. Both small localized events (ROIs 3, 4), and generalized Ca2+ oscillations (ROIs 1, 2) were seen. Movie 1 shows the spontaneous Ca2+ increases in this vessel.
Figure 3.
 
Global Ca2+ oscillations can induce retinal MVSM cell contraction. (A) The outline of a retinal arteriolar smooth muscle cell imaged before and during a global Ca2+ oscillation. (B) Changes in calcium (as F/F0) and cell area are plotted against time in the same cell.
Figure 3.
 
Global Ca2+ oscillations can induce retinal MVSM cell contraction. (A) The outline of a retinal arteriolar smooth muscle cell imaged before and during a global Ca2+ oscillation. (B) Changes in calcium (as F/F0) and cell area are plotted against time in the same cell.
Figure 4.
 
Line-scan images of Ca2+ sparks and global Ca2+ oscillations. (A) Left: shaded outline of a single retinal myocyte in an x-y Fluo-4–dyed image of a retinal vessel. The position of the transverse scan line is marked, and the resultant line-scan image (right) showing spontaneous changes in fluorescence for the selected cell is seen in the horizontal frame. Average fluorescence for this frame is plotted against time on the graph below it. A brief, localized Ca2+ spark is visible, spreading laterally from the center of the cell. (B) Transverse line-scan image from another vessel (see cell outline and scan-line position; left) in which sparks summated to give a longer lasting global Ca2+ oscillation. The gap in the line-scan image and fluorescence versus time plot results from the time taken for the image to be downloaded by the computer between acquisition cycles.
Figure 4.
 
Line-scan images of Ca2+ sparks and global Ca2+ oscillations. (A) Left: shaded outline of a single retinal myocyte in an x-y Fluo-4–dyed image of a retinal vessel. The position of the transverse scan line is marked, and the resultant line-scan image (right) showing spontaneous changes in fluorescence for the selected cell is seen in the horizontal frame. Average fluorescence for this frame is plotted against time on the graph below it. A brief, localized Ca2+ spark is visible, spreading laterally from the center of the cell. (B) Transverse line-scan image from another vessel (see cell outline and scan-line position; left) in which sparks summated to give a longer lasting global Ca2+ oscillation. The gap in the line-scan image and fluorescence versus time plot results from the time taken for the image to be downloaded by the computer between acquisition cycles.
Table 1.
 
Basic Properties of Ca2+ Sparks and Global Ca2+ Oscillations in Retinal MVSM Cells
Table 1.
 
Basic Properties of Ca2+ Sparks and Global Ca2+ Oscillations in Retinal MVSM Cells
Ca2+ Sparks n Ca2+ Sparks on Oscillations n P Oscillations n
Amplitude (ΔF/F0) 0.81 ± 0.04 102 0.69 ± 0.04 78 * 0.93 ± 0.04 162
Spread (FWHM) 1.25 ± 0.05 μm 96 1.67 ± 0.08 μm 65 , *** n/a n/a
Duration (FDHM) 23.6 ± 1.15 ms 102 22.2 ± 1.12 ms 78 NS 1992 ± 0.06 ms 162
Frequency 0.56 ± 0.06 s−1 60 cells 2.86 ± 0.25 s−1 50 cells , *** 0.13 ± 0.01 s−1 35 cells
Supplementary Materials
Movie 1 - 2.01 MB 
Spontaneous Ca2+ sparks and waves in the smooth muscle of an intact retinal arteriole (approximately 5 times actual speed). 
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Figure 1.
 
Luminal confocal section of an isolated retinal arteriole loaded for 10 minutes with the membrane-tracking dye, di-4-ANEPPS (Molecular Probes, Eugene, OR). Image dimensions = 84 μm × 98 μm.
Figure 1.
 
Luminal confocal section of an isolated retinal arteriole loaded for 10 minutes with the membrane-tracking dye, di-4-ANEPPS (Molecular Probes, Eugene, OR). Image dimensions = 84 μm × 98 μm.
Figure 2.
 
Ca2+ sparks and waves imaged in Fluo-4–loaded retinal MVSM cells. (A) Four time frames from a series of x-y images of a retinal arteriole. The MVSM cells are oriented at right angles to the long axis of the vessel. Fluorescence relative to basal values (F/F0) is represented in grayscale, as indicated on the calibration bar on the left. Four ROIs are marked, and the average fluorescence within each of these regions through the course of the 40-second experiment is plotted against time in (B). Arrows: the time point at which each frame was captured. Both small localized events (ROIs 3, 4), and generalized Ca2+ oscillations (ROIs 1, 2) were seen. Movie 1 shows the spontaneous Ca2+ increases in this vessel.
Figure 2.
 
Ca2+ sparks and waves imaged in Fluo-4–loaded retinal MVSM cells. (A) Four time frames from a series of x-y images of a retinal arteriole. The MVSM cells are oriented at right angles to the long axis of the vessel. Fluorescence relative to basal values (F/F0) is represented in grayscale, as indicated on the calibration bar on the left. Four ROIs are marked, and the average fluorescence within each of these regions through the course of the 40-second experiment is plotted against time in (B). Arrows: the time point at which each frame was captured. Both small localized events (ROIs 3, 4), and generalized Ca2+ oscillations (ROIs 1, 2) were seen. Movie 1 shows the spontaneous Ca2+ increases in this vessel.
Figure 3.
 
Global Ca2+ oscillations can induce retinal MVSM cell contraction. (A) The outline of a retinal arteriolar smooth muscle cell imaged before and during a global Ca2+ oscillation. (B) Changes in calcium (as F/F0) and cell area are plotted against time in the same cell.
Figure 3.
 
Global Ca2+ oscillations can induce retinal MVSM cell contraction. (A) The outline of a retinal arteriolar smooth muscle cell imaged before and during a global Ca2+ oscillation. (B) Changes in calcium (as F/F0) and cell area are plotted against time in the same cell.
Figure 4.
 
Line-scan images of Ca2+ sparks and global Ca2+ oscillations. (A) Left: shaded outline of a single retinal myocyte in an x-y Fluo-4–dyed image of a retinal vessel. The position of the transverse scan line is marked, and the resultant line-scan image (right) showing spontaneous changes in fluorescence for the selected cell is seen in the horizontal frame. Average fluorescence for this frame is plotted against time on the graph below it. A brief, localized Ca2+ spark is visible, spreading laterally from the center of the cell. (B) Transverse line-scan image from another vessel (see cell outline and scan-line position; left) in which sparks summated to give a longer lasting global Ca2+ oscillation. The gap in the line-scan image and fluorescence versus time plot results from the time taken for the image to be downloaded by the computer between acquisition cycles.
Figure 4.
 
Line-scan images of Ca2+ sparks and global Ca2+ oscillations. (A) Left: shaded outline of a single retinal myocyte in an x-y Fluo-4–dyed image of a retinal vessel. The position of the transverse scan line is marked, and the resultant line-scan image (right) showing spontaneous changes in fluorescence for the selected cell is seen in the horizontal frame. Average fluorescence for this frame is plotted against time on the graph below it. A brief, localized Ca2+ spark is visible, spreading laterally from the center of the cell. (B) Transverse line-scan image from another vessel (see cell outline and scan-line position; left) in which sparks summated to give a longer lasting global Ca2+ oscillation. The gap in the line-scan image and fluorescence versus time plot results from the time taken for the image to be downloaded by the computer between acquisition cycles.
Table 1.
 
Basic Properties of Ca2+ Sparks and Global Ca2+ Oscillations in Retinal MVSM Cells
Table 1.
 
Basic Properties of Ca2+ Sparks and Global Ca2+ Oscillations in Retinal MVSM Cells
Ca2+ Sparks n Ca2+ Sparks on Oscillations n P Oscillations n
Amplitude (ΔF/F0) 0.81 ± 0.04 102 0.69 ± 0.04 78 * 0.93 ± 0.04 162
Spread (FWHM) 1.25 ± 0.05 μm 96 1.67 ± 0.08 μm 65 , *** n/a n/a
Duration (FDHM) 23.6 ± 1.15 ms 102 22.2 ± 1.12 ms 78 NS 1992 ± 0.06 ms 162
Frequency 0.56 ± 0.06 s−1 60 cells 2.86 ± 0.25 s−1 50 cells , *** 0.13 ± 0.01 s−1 35 cells
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