Heterogeneous function of ryanodine receptors, but not IP3 receptors ...

Am J Physiol Heart Circ Physiol 300: H1616–H1630, 2011. First published February 25, 2011; doi:10.1152/ajpheart.00728.2010.

Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles Erika B. Westcott and William F. Jackson Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan Submitted 22 July 2010; accepted in final form 23 February 2011

Westcott EB, Jackson WF. Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles. Am J Physiol Heart Circ Physiol 300: H1616–H1630, 2011. First published February 25, 2011; doi:10.1152/ajpheart.00728.2010.—The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP3Rs in Ca2⫹ signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca2⫹ sparks and Ca2⫹ waves, which were inhibited by the RyR antagonists ryanodine (10 ␮M) or tetracaine (100 ␮M). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca2⫹ and constricted the arteries. The blockade of IP3Rs with xestospongin D (5 ␮M) or 2-aminoethoxydiphenyl borate (100 ␮M) or the inhibition of phospholipase C using U-73122 (10 ␮M) also attenuated Ca2⫹ waves without affecting Ca2⫹ sparks. Importantly, the IP3Rs and phospholipase C antagonists decreased global intracellular Ca2⫹ and dilated the arteries. In contrast, cremaster arterioles displayed only Ca2⫹ waves: Ca2⫹ sparks were not observed, and neither ryanodine (10 –50 ␮M) nor tetracaine (100 ␮M) affected either Ca2⫹ signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca2⫹ waves were attenuated by xestospongin D (5 ␮M), 2-aminoethoxydiphenyl borate (100 ␮M), and U-73122 (10 ␮M), accompanied by decreased global intracellular Ca2⫹ and vasodilation. These findings highlight the contrasting roles played by RyRs and IP3Rs in Ca2⫹ signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles. microcirculation; ion channels; vascular smooth muscle; inositol 1,4,5-trisphosphate

(SR) of vascular smooth muscle cells contains at least two types of Ca2⫹-release channels: ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) (78). RyRs underlie Ca2⫹ sparks (15), and they may also participate in more global intracellular Ca2⫹ events through Ca2⫹-induced Ca2⫹ release (CICR) (78). Calcium release during G protein-coupled receptor activation is mediated by IP3Rs, and these Ca2⫹ release channels also underlie Ca2⫹ waves (78). In some systems, both RyRs and IP3Rs contribute to Ca2⫹ signals (6, 78). What remains unclear is the role played by these channels in microvascular smooth muscle and the regulation of myogenic tone. RyR-mediated Ca2⫹ sparks have been observed in retinal arterioles (21, 22, 76) and ureter arterioles (13). In contrast, recent studies of cells isolated from first-order rat cremaster arterioles failed to detect Ca2⫹ sparks (82). However, further THE SARCOPLASMIC RETICULUM

Address for reprint requests and other correspondence: W. F. Jackson, Michigan State Univ., B420 Life Sciences, East Lansing, MI 48824 (e-mail: [email protected]). H1616

studies of ureter arteriolar smooth muscle cells (10) found no role for RyRs in these microvessels. While most studies of smooth muscle from larger vessels suggest that Ca2⫹ sparks activate sarcolemmal large-conductance Ca2⫹-activated K⫹ (BKCa) channels and contribute to the negative feedback regulation of membrane potential and vascular tone (78), studies of renal arterioles suggest that RyRs participate in the positive feedback regulation of vascular tone through CICR in this microcirculation (3, 25, 26). Thus there appear to be substantial regional differences in the function of RyRs in the microcirculation. Similarly, the role played by IP3Rs in the regulation of myogenic tone in the microcirculation is largely unknown. Recent studies of ureter arterioles suggest that IP3Rs play a dominant role in Ca2⫹ signaling in these vessels during agonist-induced constriction (10). However, the arterioles in these studies were unpressurized, hence the function of IP3Rs during pressure-induced myogenic tone was not addressed. In rat first-order cremaster arterioles, the IP3R antagonist 2-aminoethoxydiphenyl borate (2-APB) has been reported to inhibit myogenic tone (71) (consistent with a major role for IP3Rs) or have little effect (53). The lack of Ca2⫹ waves observed in small fluo-4-loaded pressurized mesenteric arteries with substantial myogenic tone (64) would appear to argue against a major role for IP3Rs in basal myogenic tone in these vessels. Thus the function of IP3Rs in arteriolar and resistance artery smooth muscle and their role in myogenic tone remain unclear. The purpose of the present study was to characterize the subsarcolemmal Ca2⫹ signals in smooth muscle cells of small skeletal muscle arterioles compared with those observed in upstream feed arteries and to test the hypothesis that RyRs and IP3Rs contribute to Ca2⫹ signals and myogenic tone in both vessel types. We found that RyRs participate in both Ca2⫹ sparks and Ca2⫹ waves in feed artery smooth muscle cells and contribute to the negative feedback regulation of myogenic tone in these small arteries. In contrast, RyRs appeared silent in smooth muscle cells of cremaster arterioles, contributing to neither Ca2⫹ sparks, Ca2⫹ waves, nor myogenic tone. On the other hand, we observed that IP3Rs and upstream phospholipase C (PLC) importantly contributed to Ca2⫹ waves, global Ca2⫹ levels, and myogenic tone in smooth muscle cells of both feed arteries and arterioles. Our findings emphasize the differences in function between RyRs and IP3Rs in feed arteries and demonstrate heterogeneity in the function of RyRs in feed arteries compared with their downstream arterioles. METHODS

Animal and vessel preparation. All experiments were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Michigan State University and were performed in accordance with the Guide for the Care and Use of

0363-6135/11 Copyright © 2011 the American Physiological Society

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CALCIUM SIGNALING IN ARTERIES AND ARTERIOLES

Laboratory Animals of the National Research Council (19a). Male golden Syrian hamsters (6 –10 wk, 75–150 g, Harlan) were euthanized by CO2 asphyxiation, followed by cervical dislocation. The right and left cremaster muscles were quickly removed and placed in 4°C Ca2⫹-free physiological salt solution (Ca2⫹-free PSS) containing (in mM) 137 NaCl, 5.6 KCl, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4, 295 mosmol/kgH2O). Second-order cremaster arterioles were isolated by hand dissection with the aid of a stereomicroscope as previously described (14, 41). Cremaster muscle feed arteries were dissected in situ by surgically exposing the iliac arteries and carefully isolating small artery branches that were upstream from the cremaster muscle microcirculation. Vessel cannulation. Arterioles and feed arteries with intact endothelium were transferred to a cannulation chamber using a 50 –100-␮l Wiretrol pipette (Drummond Scientific, Broomal, PA), cannulated onto glass micropipettes, and secured to the pipettes using 11-0 ophthalmic suture (Ashaway Line and Twine, Ashaway, RI). The chamber was then secured to the stage of a microscope (Leica DMIL, Wetzlar, Germany) where the vessels were visualized, heated to 34°C (cremaster arterioles) or 37°C (feed arteries), pressurized to 80 cmH2O and allowed to develop myogenic tone (14, 40). All vessels studied had a minimum of 20% resting myogenic tone compared with the maximum diameters of the vessels obtained in Ca2⫹-free PSS. Vessels were constantly superfused with PSS, containing (in mM) 140 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4), alone or containing a drug. Calcium imaging. The smooth muscle cells of cannulated vessels were loaded with the intensiometric Ca2⫹ indicator fluo-4 by bath incubation. The dye solution contained 5 ␮M fluo-4 AM dye (Invitrogen, Carlsbad, CA) in 0.5% dimethyl sulfoxide (DMSO) and 0.1% bovine serum albumin (USB, Cleveland, OH) in Ca2⫹-free PSS. This solution was applied to the vessels for 2 h at room temperature, followed by a 30-min superfusion with PSS to wash fluo-4 from the bath and to allow for dye deesterification and gradual temperature increase. All vessels were imaged using a long working-distance ⫻40 H2O immersion objective (numerical aperature, 0.8; and working distance, 3 mm; Leica). Fluo-4 fluorescence at 526 nm was acquired at 30 frames/s using a spinning-disc confocal system (CSU-10B, Solamere, Salt Lake City, UT) with 488 nm laser illumination (Solamere) and an intensified CCD camera (XR Mega-10, Stanford Photonics, Palo Alto, CA). Each recording period was 16.7 s and consisted of 500 1,024 ⫻ 1,024 pixel frames captured at 30 frames/s (0.17 ⫻ 0.17 ␮m per pixel on our system). The z-resolution with our confocal head (50 ␮m pinholes) and stated objective in PSS was 1.77 ␮m (see http://zeiss-campus.magnet.fsu.edu/articles/ spinningdisk/introduction.html for details). Images were recorded using Piper software (Stanford Photonics) and analyzed using SparkAn (courtesy of Drs. M. T. Nelson and A. D. Bonev, University of Vermont) and ImageJ (1) software. The occurrence of both Ca2⫹ sparks and Ca2⫹ waves was counted manually by separately visualizing each smooth muscle cell within a vessel using a masking procedure and scoring whether or not any Ca2⫹ sparks and/or waves appeared during playback of 500 frame sequences. These occurrences were then verified using SparkAn as increases in fluorescence that were at least 15% above basal levels for each cell for sparks and 20% above baseline for waves. SparkAn was also used to calculate the average frequency, amplitude (F/Fo), and full-duration half maximum (FDHM) of Ca2⫹ events recorded from cells in vessel walls. In SparkAn, a region of interest (ROI) (10 ⫻ 10 pixels) was placed at the peak of Ca2⫹ events on each cell that displayed at least one Ca2⫹ event during the recording period. Therefore, the frequency, amplitude, and FDHM represent the average rate of spark or wave occurrence, peak amplitude, or duration of Ca2⫹ events, respectively, per cell within a vessel. The spatial spread [full-width half maximum (FWHM)] of Ca2⫹ sparks and waves were determined using ImageJ. Hand-drawn ROIs were first used to calculate the prespark/wave basal Ca2⫹ level of each smooth muscle cell. FWHM was next calculated by comparing the basal intensity of each AJP-Heart Circ Physiol • VOL

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cell with plot profiles along lines drawn through the peak fluorescence of each spark or wave. Calcium transients in smooth muscle cells induced by caffeine (10 mM) were assessed using the ratiometric Ca2⫹-indicator fura-2 as previously described (14, 40). Fura-2 was excited at 340 and 380 nm using a DeltaRam X multiwavelength illuminator, and emission at 510 nm was measured using a D-104 photometer (Photon Technologies International, Birmingham, NJ). Both the illuminator and photometer were controlled using FeliX software (Photon Technologies International). Background fluorescence of cannulated arterioles was measured before loading and subtracted from the fluorescence values of the loaded vessels. Smooth muscle cells in cannulated vessels were loaded by bath application of fura-2 (5 ␮M) for 60 min, followed by a 30-min washout period. Diameter was simultaneously measured using Diamtrak software (T. O. Neild, Adelaide, Australia) as described below. To determine whether Ca2⫹ waves in hamster cremaster arterioles and hamster feed arteries were synchronous, SparkAn was used to produce a trace of Ca2⫹ activity in up to 20 cells/vessel by placing 10 ⫻ 10 pixel ROIs on each cell that exhibited at least one wave (see Fig. 1C for example). The data from each ROI were then combined by averaging the F/Fo of the ROIs across each time point to produce an average trace of global Ca2⫹ activity in the whole vessel during the recording period (see Fig. 1C). These averaged tracings were then analyzed to determine the presence of any peaks with fluorescence ⬎ 1.2 Fo. Measurement of vessel diameters. In fluo-4-loaded feed arteries and arterioles, steady-state diameters were measured asynchronously from Ca2⫹ measurements. Immediately before Ca2⫹ measurements, before or after drug administration, the microscope was focused to midplane, the vessels were briefly transilluminated with dim 586-nm light, and a sequence of images were acquired. The microscope was then refocused to the plane of interest for Ca2⫹ measurements. Internal vessel diameters were then measured using ImageJ from the transilluminated images. The diameter effects of all drugs were verified in preliminary experiments on vessels not loaded with fluo-4 to verify the time course of the drugs to ensure that steady states were achieved and that asynchronous diameter measures accurately represented the steady-state effects of the drugs. Measurements were typically made after 10 –15 min of drug exposure. In all other experiments, vessel internal diameters were continuously measured using Diamtrak software (T. O. Neild) (66), as previously reported (14, 40). Chemicals. Ryanodine was obtained from Ascent Scientific (Bristol, UK), and xestospongin D and 2-APB, from Calbiochem (San Diego, CA). Fluo-4 was obtained from Invitrogen. All other drugs and chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless noted otherwise in the text. Caffeine and tetraethyl ammonium (TEA) were dissolved directly into PSS. All other drugs were dissolved in DMSO and then diluted to their final concentrations in PSS. DMSO alone was without effect in cannulated vessels. Data analysis and statistics. The occurrence of Ca2⫹ sparks and Ca2⫹ waves are presented as proportions (number cells with at least 1 occurrence/total number of cells examined) ⫾ 95% confidence intervals, with confidence intervals calculated using QuickCalcs’ implementation of the modified Wald method (http://www.graphpad. com/quickcalcs/ConfInterval1.cfm). All other data are expressed as means ⫾ SE. Statistical significance was determined using Student’s t-tests, analyses of variance followed by Tukey’s test for post hoc comparisons of means, or Kruskall-Wallis test followed by Dunn’s multiple comparison test when data were not normally distributed or when group variances were heterogeneous. All statistical comparisons were performed at the 95% confidence level. RESULTS

Characterization of Ca2⫹ signals and myogenic tone in feed arteries and arterioles. We observed both small, local Ca2⫹ events that we have classified as Ca2⫹ sparks and larger, more global Ca2⫹ events (Ca2⫹ waves) in fluo-4-loaded feed arteries 300 • MAY 2011 •

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Fig. 1. Representative images of Ca2⫹ sparks and waves in cremaster feed arteries and arterioles. A: confocal images of fluo-4-loaded hamster feed arteries (HFAs) and hamster cremaster arterioles (HCAs) as indicated: basal Ca2⫹ levels (top) and peak Ca2⫹ levels (bottom) for a feed artery spark (position indicated by the arrow), feed artery waves, and cremaster arteriole waves (left to right). Sparks occur in a small, restricted area of smooth muscle cells, whereas waves occur in a much larger area of the cells. See text for more details. B: representative traces generated using SparkAn depicting the amplitude (F/Fo) vs. time for both a Ca2⫹ spark and a Ca2⫹ wave. Calcium sparks (dotted line) had a smaller amplitude and duration compared with Ca2⫹ waves (solid line). C: Ca2⫹ waves observed in both feed arteries (left) and arterioles (right) were asynchronous: representative traces of F/Fo from 10 cells within the same vessel during one 16.7-s recording period (top) and average of the 10 traces with no peaks ⬎ 1.2 F/Fo, recorded from either feed artery or cremaster arteriole (bottom). Similar results were obtained through analysis of 122 cells from 6 feed arteries and 98 cells from 5 arterioles.

studied at physiological temperature and pressure (Fig. 1, A and B, and Table 1). The two Ca2⫹ signals were differentiated based on their pharmacology (see Role of RyRs in feed arteries and Role of IP3Rs and PLC in feed arteries) and their spatial spread within the cells: Ca2⫹ sparks were localized to small microdomains within smooth muscle cells, whereas Ca2⫹ waves involved a more global increase in Ca2⫹ (Fig. 1, and

Table 1). Calcium waves also had a significantly longer duration and larger amplitude than Ca2⫹ sparks, as seen in the representative traces in Fig. 1B and in Table 1. Calcium waves were asynchronous and were observed throughout the recording period (Fig. 1C). The length of smooth muscle cross sections in confocal slices were measured in fluo-4-loaded vessels and compared

Table 1. Properties of Ca2⫹ signals in cremaster feed arteries and arterioles Cross-Section Length, ␮m

Feed arteries Sparks Feed arteries Waves Arterioles Waves

FWHM, ␮m

Frequency, Hz

Amplitude, F/Fo

FDHM, s

n

85 ⫾ 1.1 (63)

6 ⫾ 0.3 (63)

0.19 ⫾ 0.02

1.46 ⫾ 0.02

0.56 ⫾ 0.02

21–23

85 ⫾ 1.0 (63)

57 ⫾ 1.4* (63)

0.39 ⫾ 0.05*

1.68 ⫾ 0.02*

1.12 ⫾ 0.08*

21–23

34 ⫾ 0.5*† (87)

25 ⫾ 0.5*† (87)

0.39 ⫾ 0.05*

1.66 ⫾ 0.05*

1.94 ⫾ 0.33*

29

Values are means ⫾ SE; N, number of cells; n, number of vessels. FWHM, full-width, half maximum; FDHM, full-duration, half maximum. For cross-section length and FWHM, measurements were made on 3 cells that displayed Ca2⫹ events per vessel. See text for details. *P ⬍ 0.05 compared with feed artery Ca2⫹ sparks; †P ⬍ 0.05 compared with feed artery. AJP-Heart Circ Physiol • VOL

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with the full-width, half maxima (FWHM) of each Ca2⫹ event. The average smooth muscle cell cross-section length was 85 ⫾ 1.0 ␮m (n ⫽ 63 cells from 21 vessels) in feed arteries. Calcium sparks had FWHM that were considerably smaller than crosssection lengths (6.5 ⫾ 0.02%, n ⫽ 63 cells from 21 vessels), whereas Ca2⫹ waves had FWHM that were much closer to the entire cross-section length (68 ⫾ 2%, n ⫽ 63 cells from 21 vessels). Calcium sparks significantly differed in frequency, amplitude, and duration (FDHM) compared with waves in feed arteries (Table 1). Calcium sparks were observed in 32% of feed artery smooth muscle cells (95% confidence interval ⫽ 29 –36%; 531 cells from 23 vessels), whereas Ca2⫹ waves were observed in 55% of cells (95% confidence intervals ⫽ 51–59%, 585 cells from 23 vessels, P ⬍ 0.05). In contrast to our observations in feed arteries and prior studies in other larger vessels, Ca2⫹ sparks were not observed in the smooth muscle cells of fluo-4-loaded cremaster arterioles: 0 cells displayed Ca2⫹ sparks out of 586 cells examined in 29 arterioles. Calcium waves, however, were routinely observed in pressurized arterioles: 321 cells displayed Ca2⫹ waves out of 586 examined in 29 arterioles (55%; 95% confidence interval ⫽ 51–59%). The frequency, amplitude, and FDHM of Ca2⫹ waves were similar to those observed in feed arteries (Table 1). However, the FWHM of the Ca2⫹ waves in arteriolar smooth muscle cells was less than that of feed arteries (25 ⫾ 0.5 ␮m, n ⫽ 87 cells from 29 vessels). This is consistent with the smaller size of these vessels (average cross-section lengths of confocal slices in the arterioles ⫽ 34 ⫾ 0.5 ␮m; P ⬍ 0.05 compared with feed arteries, Table 1), as the arteriolar Ca2⫹ waves occupied 75 ⫾ 1% of slice length, greater than the fraction observed in feed arteries (P ⬍ 0.05). Importantly, Ca2⫹ waves in arterioles were much larger than

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Ca2⫹ sparks observed in the feed arteries (Table 1). Like the Ca2⫹ waves observed in feed arteries, arteriolar Ca2⫹ waves were asynchronous and observed throughout the recording period (Fig. 1C). Effects of SR Ca2⫹ depletion in feed arteries and arterioles. To verify that the Ca2⫹ signals observed in feed arteries and arterioles originated from intracellular Ca2⫹ stores, we examined the effects of the smooth sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA) inhibitor thapsigargin (100 nM) (58) in fluo-4-loaded feed arteries and arterioles (Fig. 2). This SERCA inhibitor abolished Ca2⫹ sparks in feed arteries. Before the application of thapsigargin (100 nM), Ca2⫹ sparks were observed in 43 of 188 smooth muscle cells (22.9%; 95% confidence interval ⫽ 17.4 –29.4; n ⫽ 3 feed arteries) with mean amplitude of 1.63 ⫾ 0.02 F/Fo and frequency of 0.1 ⫾ 0.01 Hz. In the presence of thapsigargin, no Ca2⫹ sparks were observed in any cells (0 of 201 cells, n ⫽ 3 arteries; P ⬍ 0.05). In both feed arteries and arterioles, the inhibition of SERCA nearly eliminated the occurrence of Ca2⫹ waves (Fig. 2, A and D). Thapsigargin (100 nM) also significantly decreased the amplitude and frequency of the few remaining Ca2⫹ waves in both feed arteries (Fig. 2A) and arterioles (Fig. 2D). In both classes of vessel, thapsigargin (100 nM) produced a significant increase in the global fluo-4 signal (an index of global Ca2⫹ levels) (Fig. 2, B and E) and vasoconstriction (Fig. 2, C and F). The removal of extracellular Ca2⫹ after exposure to thapsigargin decreased the global fluo-4 signal to 60 ⫾ 2 and 59 ⫾ 3% of values in PSS and dilated the vessels from 158 ⫾ 7 to 226 ⫾ 8 ␮m and from 38 ⫾ 2 to 77 ⫾ 3 ␮m (see dashed lines in Fig. 2, C and F) in feed arteries and arterioles, respectively (n ⫽ 3 for each). These data suggest that the thapsigargin-

Fig. 2. Store depletion inhibits Ca2⫹ waves but increases global Ca2⫹ and myogenic tone in both feed arteries and arterioles. Data are means ⫾ 95% confidence intervals for occurrence (Occ) of Ca2⫹ waves (A and D). All other data are means ⫾ SE for amplitude (Amp) and frequency (Freq) of Ca2⫹ waves (A and D); global fluo-4 intensity, an index of global intracellular Ca2⫹ (B and E); and diameter (C and F) in the absence [physiological salt solution (PSS)] or presence of the sarco(endo)plasmic reticulum Ca2⫹-ATPase inhibitor thapsigargin (100 nM) as indicated. Rel, relative units. *P ⬍ 0.05 compared with value in PSS, n ⫽ 3. Dashed lines in C and F represent the maximum diameters of the vessels in 0 Ca2⫹ PSS.

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induced changes in global Ca2⫹ and diameter largely resulted from the stimulation of Ca2⫹ influx. Effects of intravascular pressure on myogenic tone and Ca2⫹ signals. The effects of different intravascular pressures on Ca2⫹ signals and myogenic tone were also characterized in both feed arteries and arterioles (Figs. 3 and 4). The diameter of both vessel types similarly responded to changes in intravascular pressure in the presence and absence of Ca2⫹ (Fig. 3). In the feed arteries, increases in intravascular pressure had no significant effect on the occurrence, amplitude, or frequency of Ca2⫹ sparks (Fig. 4). In contrast, changes in intravascular pressure had comparable effects on Ca2⫹ waves in feed arteries and arterioles: Ca2⫹ wave occurrences were increased at 40, 80, and 120 compared with 20 cmH2O, Ca2⫹ wave amplitudes were unaltered by the pressure changes, and Ca2⫹ wave frequency increased at 80 and 120 compared with 20 and 40 cmH2O (Fig. 4). Calcium sparks were not observed in arterioles at any pressure. Role of RyRs in feed arteries. Superfusion of feed arteries with the RyR antagonist ryanodine (10 ␮M) abolished the occurrence of Ca2⫹ sparks: 0 cells out of 118 examined from 6 feed arteries displayed these events (P ⬍ 0.05). Ryanodine (10 ␮M) also significantly reduced the number of smooth muscle cells that displayed Ca2⫹ waves to 13% (95% confidence interval 7–21%, P ⬍ 0.05, n ⫽ 6 vessels) (Fig. 5, A and B). The application of ryanodine also led to a significant increase in global fluo-4 intensity, suggesting an increase in intracellular Ca2⫹ (Fig. 5C) and significant vasoconstriction (Fig. 5D). Because ryanodine may deplete SR Ca2⫹ stores in some systems (80), the experiments were repeated using tetracaine (100 ␮M), which blocks RyRs without depleting SR Ca2⫹ stores (22). Similar results were obtained (Fig. 5, E–H): both Ca2⫹ sparks and waves were inhibited, associated with a global increase

Fig. 3. Feed artery and arterioles display similar pressure-diameter relationships. Data are means ⫾ SE (n ⫽ 3) for feed artery (A) and arteriole (B) diameters at 20, 40, 80, and 120 cmH2O in Ca2⫹-containing PSS (Œ) and Ca2⫹-free PSS (). AJP-Heart Circ Physiol • VOL

in Ca2⫹ and vasoconstriction. The ryanodine- and tetracaineinduced increases in global Ca2⫹ and the associated vasoconstrictions are consistent with the negative feedback role that has been reported for RyRs in other systems (78). To further test the hypothesis that RyRs participate in the negative feedback regulation of feed artery tone, we compared the vasomotor effects of ryanodine with those of the BKCa channel blockers paxilline (100 nM) (51) or TEA at a concentration of TEA (1 mM) that we have previously shown to selectively inhibit BKCa channels in hamster vascular smooth muscle (11, 12). We verified that paxilline (100 nM) and TEA (1 mM) were equieffective on feed artery diameter. From a resting diameter of 155.4 ⫾ 3.8 ␮m, paxilline (100 nM) constricted feed arteries by 37.7 ⫾ 3.9 ␮m. In the same vessels, TEA (1 mM) produced similar constriction of 43.8 ⫾ 6.3 ␮m (n ⫽ 4, P ⬎ 0.05). Thus the concentrations of the BKCa channel blockers used appeared to have equivalent effects in these vessels. Alone, both paxilline (Fig. 6A) and ryanodine (Fig. 6, B, D, and E) caused significant constriction of feed arteries. However, in the presence of paxilline (100 nM), ryanodine (10 ␮M) caused no additional constriction (Fig. 6A), consistent with published studies implicating the functional coupling of RyRs and BKCa channels (78). If the order of application of the drugs were reversed, ryanodine (10 ␮M) only attenuated the paxilline-induced constriction of feed arteries (Fig. 6B). Identical results were obtained with TEA (1 mM) (Fig. 6, C and D). These data suggest that while the activity of RyRs may be functionally linked to BKCa channels, they are not the only regulators of BKCa channel activity. To verify that it was not simply the constriction induced by paxilline (100 nM) or TEA (1 mM) that inhibited responses to ryanodine (10 ␮M) (Fig. 6, A and C), the effects of this RyR antagonist were examined in the absence (Fig. 6E) and presence of a similar level of constriction induced by the L-type Ca2⫹ channel agonist Bay K 8644 (5 nM) (Fig. 6F). As can be seen in Fig. 6F, ryanodine (10 ␮M) still was able to induce vasoconstriction in the Bay K 8644 preconstricted arteries. These data suggest that the loss of response to ryanodine that was observed in the presence of paxilline (100 nM) or TEA (1 mM) (Fig. 6, A and C) was not simply due to the constriction induced by these BKCa channel blockers. The effects of TEA were also examined in fluo-4-loaded feed arteries (Fig. 7, A–C). In contrast to the effects of ryanodine (10 ␮M), the BKCa channel blocker increased the occurrence, amplitude, and frequency of Ca2⫹ waves, while increasing global Ca2⫹ and constricting the arteries (Fig. 7, A–C). The increase in occurrence and frequency of Ca2⫹ waves prevented an accurate quantitation of feed artery Ca2⫹ spark activity in the presence of TEA. Lack of a role for RyRs in arterioles. Consistent with the absence of Ca2⫹ sparks in smooth muscle cells of cremaster arterioles, we found that ryanodine (10 ␮M) had no significant effect on Ca2⫹ signals in these microvessels: in the presence of ryanodine, Ca2⫹ waves were still observed (n ⫽ 10, P ⬎ 0.05). To rule out the possibility that the concentration of ryanodine used was insufficient, we repeated these experiments using a higher concentration of this alkaloid (50 ␮M) and obtained similar results: ryanodine (50 ␮M) had no effect on Ca2⫹ wave occurrence, amplitude, or frequency (Fig. 8, A–C). Consistent with the lack of effect of ryanodine on Ca2⫹ waves, this 300 • MAY 2011 •

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Fig. 4. Effect of changes in intravascular pressure on the occurrence (top), amplitude (middle), and frequency (bottom) of Ca2⫹ sparks (left) and Ca2⫹ waves (right) for both feed arteries and arterioles as indicated. At pressures of 20, 40, 80, and 120 cmH2O, there were no differences in the occurrence, amplitude, or frequency of Ca2⫹ waves between feed arteries and arterioles (right); P ⬎ 0.05. *P ⬍ 0.05, significantly different from the equivalent value at an intravascular pressure of 20 cmH2O; n ⫽ 3 feed arteries or arterioles.

alkaloid also was without significant effect on global intracellular Ca2⫹ (Fig. 8B) and arteriolar diameter (Fig. 8C). Tetracaine (100 ␮M, Fig. 8, D–F), another RyR antagonist, was also without significant effect on any of the parameters measured. Functional evidence for expression of RyRs in arterioles and efficacy of ryanodine. Because ryanodine had no effect in the arterioles, we examined the functional expression of RyRs

using the RyR agonist caffeine (78) (10 mM, Fig. 9) and the ability of ryanodine to block the effects of caffeine as a test of the efficacy of ryanodine (Fig. 9, C and D). Similar to Hill et al. (34), we found that caffeine had biphasic effects on both intracellular Ca2⫹ and vessel diameter in arterioles pressurized to 80 cmH2O that were difficult to interpret (Fig. 9A). However, vessels pressurized to 20 cmH2O, with little myogenic

Fig. 5. Ryanodine receptors contribute to Ca2⫹ sparks, Ca2⫹ waves, and myogenic tone in cremaster feed arteries. A–D: data for vessels in the presence and absence of ryanodine (10 ␮M). E–H: data for vessels in the presence and absence of tetracaine (100 ␮M). Data are means ⫾ 95% confidence intervals for occurrence of Ca2⫹ sparks and waves (A, B, E, and F) or means ⫾ SE for Ca2⫹ wave amplitude (F/Fo); frequency (in Hz); global fluo-4 signal, an index of global intracellular Ca2⫹(C and G); and diameter (D and H) in fluo-4-loaded cremaster feed arteries at 80 cmH2O in the absence (PSS) and presence of ryanodine (10 ␮M, n ⫽ 6) or tetracaine (100 ␮M, n ⫽ 3) as indicated. *P ⬍ 0.05, significantly different from value in PSS. Maximum diameter of arteries is shown by dashed line in D and H.


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Fig. 6. Blockade of large-conductance Ca2⫹-activated K⫹ (BKCa) channels inhibits ryanodine-induced constriction of feed arteries. Data are mean diameters ⫾ SE in the absence (PSS) or presence of the BKCa channel blockers paxilline (100 nM, n ⫽ 4; A and B) or tetraethyl ammonium (TEA, 1 mM, n ⫽ 6; C); the ryanodine receptor antagonist ryanodine (10 ␮M; A–F); the voltage-gated Ca2⫹ channel agonist Bay K 8644 (5 nM, n ⫽ 6; F); or combinations of paxilline ⫹ ryanodine (A and B), TEA ⫹ ryanodine (C and D), or Bay K 8644 ⫹ ryanodine (F) as indicated. *P ⬍ 0.05, significantly different from PSS. ␺P ⬍ 0.05, significantly different from PSS, but not significantly different from adjacent group (P ⬎ 0.05). **P ⬍ 0.05, significantly different from PSS and adjacent group.

tone, consistently responded to caffeine (10 mM) with a typical, transient increase in intracellular Ca2⫹ that was associated with a transient constriction (Fig. 9B, n ⫽ 7, P ⬍ 0.05). In separate experiments, we found that ryanodine (10 or 50 ␮M) abolished the caffeine-induced constriction (Fig. 9, C and D), confirming that ryanodine effectively blocks RyRs in the arterioles. As above, ryanodine alone did not affect resting arteriolar diameter (Fig. 9, C and D). Consistent with the lack of effect of ryanodine on Ca2⫹ signals or diameter, ryanodine also had no effect on responses induced by the BKCa channel blockers paxilline (100 nM, Fig. 10, A and B) or TEA (1 mM) (Fig. 10, B and C), suggesting that RyRs and BKCa channels are not functionally coupled in arterioles. Thus, in contrast to our findings in feed arteries, RyRs appeared to be silent in arteriolar smooth muscle cells, contributing to neither Ca2⫹ signals nor myogenic tone in arterioles under the conditions of our experiments. Similar to the effects of TEA on Ca2⫹ waves in feed arteries, this BKCa channel blocker significantly increased the occurrence of Ca2⫹ waves in arterioles (Fig. 7D). There was a tendency for increased amplitude and frequency of Ca2⫹ waves in the presence of TEA, but these did not attain statistical significance. However, the global fluo-4 intensity was signifiAJP-Heart Circ Physiol • VOL

cantly increased (Fig. 7E), accompanied by a significant decrease in diameter (Fig. 7F) as reported above. Role of IP3Rs and PLC in feed arteries. We next investigated the role played by IP3Rs in the generation of Ca2⫹ signals and myogenic tone in feed artery smooth muscle cells. Similar to the effects of ryanodine on Ca2⫹ waves in these vessels, the IP3R antagonist xestospongin D (5 ␮M) nearly abolished the occurrence of Ca2⫹ waves and significantly decreased the amplitude and frequency of what few waves remained (Fig. 11B). Similar results were obtained using 2-APB (100 ␮M) (Fig. 11F), another putative IP3R antagonist. Consistent with a role for IP3 and IP3Rs in the mechanisms underlying Ca2⫹ waves, we also found that an inhibitor of PLC, U-73122 (10 ␮M), greatly attenuated the occurrence, amplitude, and frequency of Ca2⫹ waves in feed artery smooth muscle cells (Fig. 11J). The inactive analog of this compound (U-73343, 10 ␮M) was without effect: Ca2⫹ spark and Ca2⫹ wave occurrence, amplitude, and frequency were not significantly different from values in PSS, and global Ca2⫹ and vessel diameter were also unaffected by U-73343 (P ⬎ 0.05, n ⫽ 6). However, in distinct contrast to the effects of ryanodine on Ca2⫹ sparks, neither the IP3R antagonists (xestospongin D or 2-APB) nor the PLC inhibitor (U-73122) had any effect on 300 • MAY 2011 •

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Fig. 7. Blockade of BKCa channels increases Ca2⫹ waves, global Ca2⫹, and myogenic tone in feed arteries and arterioles. Data are means ⫾ 95% confidence intervals for Ca2⫹ wave occurrence (A and D) or means ⫾ SE for Ca2⫹ wave amplitude (F/Fo) or frequency (in Hz; A and D); global fluo-4 signal, an index of global intracellular Ca2⫹ (B and E); or diameter (C and F) in the absence or presence of the BKCa channel blocker TEA (1 mM) as indicated. *P ⬍ 0.05, significantly different from equivalent value in PSS; n ⫽ 3 for feed arteries, and n ⫽ 6 for arterioles.

the occurrence, amplitude, or frequency of Ca2⫹ sparks in feed artery smooth muscle cells (Fig. 11, A, E, and I). Similarly, the spatial spread (FWHM) and duration (FDHM) of Ca2⫹ sparks were not significantly affected by xestospongin D, 2-APB, or U-73122 (Table 2).

In contrast to the effects of ryanodine or tetracaine on global Ca2⫹ and diameter in feed arteries, xestospongin D, 2-APB, or U-73122 each reduced global Ca2⫹ signals and dilated the feed arteries (Fig. 11, C and D, G and H, and K and L). The dramatic effects of the IP3R antagonists in feed arteries indicate that

Fig. 8. Ryanodine receptors do not contribute to Ca2⫹ signals or myogenic tone in cremaster arterioles. In fluo-4-loaded, cannulated arterioles at 80 cmH2O, Ca2⫹ waves, but not Ca2⫹ sparks, were routinely observed. Data are means ⫾ 95% confidence intervals for Ca2⫹ wave occurrence (A and D) or means ⫾ SE for Ca2⫹ wave amplitude (F/Fo), Ca2⫹ wave frequency (in Hz) (A and D); global fluo-4 intensity, an index of global intracellular Ca2⫹ (B and E); and diameter (C and F) in the absence (PSS) or presence of the ryanodine receptor antagonists ryanodine (50 ␮M, n ⫽ 6; A–C) or tetracaine (100 ␮M, n ⫽ 3; D–F) as indicated. No significant differences for PSS vs. values in the presence of ryanodine receptor antagonists were observed (P ⬎ 0.05).


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CALCIUM SIGNALING IN ARTERIES AND ARTERIOLES DISCUSSION

Fig. 9. Functional evidence for ryanodine (Ryd) receptors in cremaster arterioles. A: typical record of diameter (left-axis, solid trace) and fura-2 ratio of emission intensity for 340 nm/380 nm illumination (right axis, dotted trace) in response to 10 mM caffeine (heavy black line) in an arteriole pressurized to 80 cmH2O. Data shown are normalized to baseline values for comparison to B. Application of 10 mM caffeine caused a biphasic response: a transient increase in the fura-2 ratio and decrease in diameter superimposed on a slow, but long-lasting, fall in the fura-2 ratio and dilation. Similar results were obtained in 6 additional arterioles. B–D: data are means ⫾ SE. B: diameters (normalized to precaffeine values, left axis, top trace) and fura- 2 ratio (normalized to precaffeine baseline, right axis, bottom trace) in arterioles pressurized to 20 cmH2O. Heavy solid line represents the time of exposure to caffeine (10 mM) as indicated; n ⫽ 7. C and D: caffeine (10 mM) and constricted arterioles, and this constriction was blocked in the presence of ryanodine (C, 10 ␮M, n ⫽ 8; and D, 50 ␮M, n ⫽ 7) as indicated. *P ⬍ 0.05, significantly different from PSS.

unlike RyRs, IP3Rs are not involved in the negative feedback regulation of myogenic tone in these small arteries. Role of IP3Rs in arteriolar smooth muscle. Similar to our observations in feed arteries, we found that antagonists of IP3Rs (xestospongin D or 2-APB) and an inhibitor of PLC (U-73122) nearly abolished the occurrence of Ca2⫹ waves in cremaster arterioles and significantly decreased the amplitude of the remaining waves (Fig. 12, A, D, and G). Both 2-APB and U-73122 also significantly reduced the frequency of the residual waves (Fig. 12, D and G). Xestospongin D tended to reduce the Ca2⫹ wave frequency, but this did not attain statistical significance (P ⫽ 0.07; Fig. 12A). As was observed in feed arteries, these antagonists also reduced global Ca2⫹ levels and dilated the arterioles (Fig. 12, B and C, E and F, and H and I). AJP-Heart Circ Physiol • VOL

Our studies highlight that significant differences exist in the role played by RyRs in regulating both subsarcolemmal Ca2⫹ signals and myogenic tone in feed arteries and their downstream arterioles and show that RyRs and IP3Rs serve different roles in regulating myogenic tone. We found that RyRs underlie Ca2⫹ sparks, contribute to Ca2⫹ waves, and participate in the negative feedback regulation of myogenic tone in feed arteries. However, RyRs appear to be silent in cremaster arterioles and contribute neither to the regulation of smooth muscle Ca2⫹ signals nor myogenic tone in these microvessels. In contrast, IP3Rs importantly contribute to Ca2⫹ waves and myogenic tone, likely through a positive feedback mechanism in both feed arteries and their downstream arterioles. Prior studies have characterized the morphology and kinetics of Ca2⫹ sparks in smooth muscle cells from a number of vascular beds. The majority of studies have focused on isolated cells, and nearly all of the studies looked at smooth muscle cells from the cerebral (8, 24, 28, 43, 56, 67, 74, 77, 85), mesenteric (72, 86, 87), or pulmonary (73, 81, 84, 87) vasculatures. Across all of these studies, a wide range of spark properties have been observed: frequencies ranged 0.3–2.85 Hz, amplitudes ranged 1.2–3.2 (F/Fo), FWHM ranged 0.85– 4.03 ␮m, and FDHM ranged 13.3–190 ms. Significantly fewer studies have investigated the properties of sparks in intact vessels, and most of these have focused on vessels that were unpressurized, either without myogenic tone (21, 22, 28, 45, 48) or with tone artificially generated with a high K⫹ solution (16, 79). In these studies, the spark properties also varied: frequency ranged 0.12–1.53 Hz, amplitude ranged 1.36 –1.93 (F/Fo), and FDHM ranged 27– 60.7 ms. Only two of these studies looked at FWHM and observed spatial spreads of 1.25 (21) and 1.59 (22) ␮m. Four studies, in cerebral (33, 61) or mesenteric (54, 64) vessels, have investigated Ca2⫹ spark properties in intact, pressurized vessels, and three of these studies used vessels that had significant myogenic tone (33, 61, 64). Three of the above studies also measured only the frequency of Ca2⫹ sparks, making comparisons between the studies difficult. Thus there appears to be considerable variability in Ca2⫹ spark morphology and kinetics. In hamster cremaster feed arteries, we observed localized Ca2⫹ signals that were inhibited by the RyR antagonists ryanodine (10 ␮m) and tetracaine (100 ␮m) and the SERCA inhibitor thapsigargin (100 nM) but unaffected by the IP3R antagonists xestospongin D (5 ␮M) or 2-APB (100 ␮M) or the PLC antagonist U-73122 (10 ␮M). Based on their small spatial spread (Table 1) and this pharmacology, we have classified these signals as Ca2⫹ sparks. The amplitude of these Ca2⫹ sparks appear within the range of values reported in other systems (see preceding paragraph). However, the spatial spread (FWHM) and the duration (FDHM) that we report appear larger than what has been observed in other systems (see preceding paragraph). We do not have an explanation for this discrepancy. All of the studies outlined above used vessels or isolated cells from different vascular beds and different species of animals, and experimental conditions (temperature, pressure, solutions, etc.) varied significantly between the studies. Therefore, it is difficult to determine whether the differences in spark characteristics that we report are species related, vascular bed related, or simply due to differences in the tools used to 300 • MAY 2011 •

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Fig. 10. Ryanodine has no effect on constriction induced by blockade of BKCa channels in cremaster arterioles. Data are mean diameters ⫾ SE in the absence (PSS) or presence of the BKCa channel blockers paxilline (100 nM, n ⫽ 4; A and B) or TEA (1 mM, n ⫽ 6; C and D), the ryanodine receptor antagonist ryanodine (10 ␮M, A–D), or combinations of paxilline ⫹ ryanodine (A and B) or TEA ⫹ ryanodine (C and D) as indicated. *P ⬍ 0.05, significantly different from PSS. ␺P ⬍ 0.05, significantly different from PSS, but not significantly different from adjacent group (P ⬎ 0.05).

study these cells. It is likely that a combination of all these factors contributed to the apparent heterogeneity, and more work needs to be done, especially in intact vessels, to clearly define the properties of Ca2⫹ sparks in native vascular smooth muscle cells. Under control conditions, both feed arteries and arterioles produced asynchronous Ca2⫹ waves, whereas only feed arteries produced Ca2⫹ sparks. These differences are not the result of nonequivalent intravascular pressures, as four different pressure steps caused similar changes in myogenic tone and Ca2⫹ signals in both vessel types. Our data are consistent with recent studies in cerebral arteries by Mufti et al. (65), suggesting that increases in intravascular pressure have a greater effect on the occurrence and frequency of asynchronous Ca2⫹ waves at pressures steps below 80 cmH2O. However, our studies differ from those by Jaggar (42) who found that increases in intravascular pressure led to an increase in Ca2⫹ spark frequency in cerebral artery smooth muscle, whereas pressure had no effect on the occurrence, amplitude, or frequency of Ca2⫹ sparks in our studies over a similar range of pressures. In feed arteries, we found that the RyR antagonists ryanodine (10 ␮M) and tetracaine (100 ␮M) and the SERCA antagonist thapsigargin (100 nM) inhibited both Ca2⫹ sparks and Ca2⫹ waves, led to an elevation in global Ca2⫹, and produced vasoconstriction. These data are consistent with a number of prior studies and further support the hypothesis that RyRs, likely through their functional coupling to BKCa channels, participate in the negative feedback regulation of myogenic tone in resistance arteries (27, 52, 68). Consistent with this hypothesis, we found that ryanodine-induced constriction of feed arteries was eliminated in the presence of the BKCa channel blockers paxilline (100 nM) or TEA (1 mM). Thus we think that in feed arteries, the loss of RyR function due to block of RyRs (ryanodine or tetracaine) or depletion of Ca2⫹ stores AJP-Heart Circ Physiol • VOL

(thapsigargin) leads to decreased BKCa channel activity, membrane depolarization, and increased Ca2⫹ influx through voltage-gated Ca2⫹ channels as has been previously postulated (68). A portion of the Ca2⫹ increase and vasoconstriction induced by thapsigargin (100 nM) likely results from Ca2⫹ store depletion-induced cation entry contributing to the depolarization and Ca2⫹ increase as previously shown in first-order rat cremaster arterioles (71). In contrast to the effects of the BKCa channel blockade on responses to ryanodine, ryanodine only blunted paxilline- or TEA-induced vasoconstriction in feed arteries. These data suggest that Ca2⫹ release through RyRs only provides a fraction of the signal responsible for activity of BKCa channels and that Ca2⫹ from other sources likely contributes to the regulation of BKCa channels in hamster feed arteries. Our observation that ryanodine or tetracaine also inhibit Ca2⫹ waves in feed arteries supports prior studies in several smooth muscles (19, 29, 32, 37), including retinal arteriolar smooth muscle cells (76), where RyRs contribute to Ca2⫹ waves, likely through CICR. Our studies differ from reports in renal afferent arterioles where ryanodine dilates the arterioles and reduces agonist-induced vasoconstriction (3). These data support our contention that there is significant regional heterogeneity in the function of RyRs in vascular smooth muscle cells. It is also possible that the inhibitory effect of ryanodine (10 ␮M) on Ca2⫹ waves in feed arteries was due to ryanodineinduced depletion of intracellular stores (36, 38) or other effects of ryanodine or tetracaine on IP3R-mediated Ca2⫹ signals (59). However, we found that Ca2⫹ waves in secondorder cremaster arterioles were unaffected by ryanodine (10–50 ␮M) or tetracaine (100 ␮M), suggesting that such off-target effects are unlikely. Further studies will be required to determine the precise role played by RyRs in Ca2⫹ wave genesis in feed arteries. 300 • MAY 2011 •

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Fig. 11. Inositol 1,4,5-trisphosphate (IP3) receptors and phospholipase C contribute to Ca2⫹ waves and myogenic tone but not Ca2⫹ sparks in cremaster feed arteries. Data are means ⫾ 95% confidence intervals for occurrence of Ca2⫹ sparks (A, E, and I) or Ca2⫹ waves (B, F, and J). All other data are means ⫾ SE for amplitude (F/Fo), and frequency (in Hz) of Ca2⫹ sparks (A, E, and I) and Ca2⫹ waves (B, F, and J); global fluo-4 intensity, an index of global intracellular Ca2⫹ (C, G, and K); and diameter (D, H, and L) in the absence (PSS) or presence of the IP3 receptor antagonists xestospongin D (5 ␮M, n ⫽ 5; A–D) or 2-aminoethoxydiphenyl borate (2-APB, 100 ␮M, n ⫽ 6; E–H); or the phospholipase C antagonist U-73122 (10 ␮M, n ⫽ 6; I–L). *P ⬍ 0.05, significantly different from value in PSS; n ⫽ 6. Dashed lines in D, H, and L represent the maximum diameters of the vessels in 0 Ca2⫹ PSS.

In contrast to our findings in the feed arteries, we were unable to detect Ca2⫹ sparks in cremaster arterioles using identical methods. Supporting these observations, we found that the RyR antagonists ryanodine (10 –50 ␮M) or tetracaine (100 ␮M) had no effect on Ca2⫹ wave dynamics, global intracellular Ca2⫹, myogenic tone, or constriction induced by the BKCa channel blockers paxilline (100 nM) or TEA (1 mM) in pressurized second-order cremaster arterioles. These data suggest that RyRs do not participate in Ca2⫹ signaling underlying myogenic tone in second-order skeletal muscle arterioles, at least under the conditions of our experiments. We cannot exclude the possibility that we may have missed some population of short-duration, low-amplitude events mediated by RyRs in the arterioles. However, if such events were present, then they were not coupled to other Ca2⫹ signals or to the regulation of myogenic tone in these microvessels, because neither ryanodine (10 –50 ␮M) nor tetracaine (100 ␮M) produced any significant effects in the arterioles. The lack of effect of ryanodine (10 –50 ␮M) and tetracaine (100 ␮M) in the arterioles, despite the presence of apparently functional RyRs, as assessed by caffeine-induced Ca2⫹ transients and constriction, also suggests that the effects of ryanodine and tetracaine observed in feed arteries are not simply off-target as noted above. AJP-Heart Circ Physiol • VOL

Our findings of no Ca2⫹ sparks in smooth muscle cells of second-order cremasteric arterioles are in agreement with recent studies on smooth muscle cells isolated from first-order rat cremasteric arterioles by Yang et al. (82), who also failed to detect Ca2⫹ sparks in their experiments, and recent observations in precapillary arterioles in ureter and vas deferens (10). However, in contrast to the study by Yang et al. (82), who found that ryanodine significantly constricted first-order rat cremaster arterioles studied via pressure myography, we found that neither ryanodine (10 –50 ␮M) nor tetracaine (100 ␮M) had any significant effect on diameter or intracellular Ca2⫹ signals in second-order hamster cremaster arterioles. This difference between arteriolar branch order may mean that firstorder arterioles are transitional between feed arteries, where we found that ryanodine or tetracaine inhibited Ca2⫹ sparks and waves and produced robust vasoconstriction, and second-order arterioles, where RyRs appeared to be silent. We do not think that our data can be explained by a complete lack of RyRs or a lack of efficacy of ryanodine in the arterioles. Caffeine produced robust Ca2⫹ transients in arteriolar smooth muscle cells, demonstrating the functional presence of RyRs. Furthermore, we were able to block the effects of caffeine with ryanodine (10 –50 ␮M), verifying that the concentrations of this alkaloid that we used effectively inhibited RyRs. Thus our 300 • MAY 2011 •

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Table 2. Lack of effects of xestospongin D, 2-APB, or U-73122 on Ca2⫹ spark properties in feed arteries Treatment

FWHM, ␮m

FDHM, s

n

PSS Xestospongin-D (5 ␮M) 2-APB (100 ␮M) U-73122 (10 ␮M)

5.6 ⫾ 0.4 (30) 5.0 ⫾ 0.4 (15) 5.4 ⫾ 0.4 (15) 5.2 ⫾ 0.3 (15)

0.49 ⫾ 0.06 0.39 ⫾ 0.04 0.34 ⫾ 0.07 0.41 ⫾ 0.07

10 to 11 5 5 to 6 5 to 6

Values are means ⫾ SE; N, number of cells; n, number of vessels. 2-APB, 2-aminoethoxydiphenyl borate; PSS, physiological saline solution. For FWHM, measurements were made on 3 cells that displayed Ca2⫹ events per vessel. P ⬎ 0.05, no statistically significant differences were detected between FWHM or FDHM values compared with values in PSS. See text for details.

data suggest that RyRs play little role in the regulation of basal myogenic tone in second-order hamster arterioles in contrast to the negative feedback role reported in arterial smooth muscle (44, 52, 68, 83). Our findings are also in contrast to observations made in rat retinal arterioles, where RyR-based Ca2⫹ sparks have been previously reported (21, 22) and appear to contribute to global Ca2⫹ signaling (76). RyRs also appear to contribute to positive-feedback CICR in rat renal preglomerular arterioles (25, 26). Taken with our results, these data from retinal and renal arterioles support the hypothesis that there are substantial regional differences in the function of RyRs in arteriolar smooth muscle cells, as well as differences in RyR function between arterioles and upstream feed arteries. The lack of effect of ryanodine (10 –50 ␮M) and tetracaine (100 ␮M) on Ca2⫹ signals, myogenic tone, and paxilline- or TEA-induced constriction in second-order arterioles also suggests that the source of Ca2⫹ responsible for the regulation of BKCa channels in these microvessels is likely different from what has been reported in arterial smooth muscle, where Ca2⫹ sparks importantly control BKCa channel function and smooth muscle excitability (27, 44, 52, 68). Studies in neurons (5, 30, 75) and coronary smooth muscle (31) have suggested that Ca2⫹ influx through voltage-gated Ca2⫹ channels may also regulate BKCa channels. Our preliminary studies in cremaster arterioles are consistent with this hypothesis (7). Further studies will be required to critically test this hypothesis in the microcirculation. Our studies do not illuminate the mechanisms responsible for the differences in function of RyRs between vessels. However, prior studies in other smooth muscles have suggested that the pattern of RyR isoform expression can significantly alter the function of RyRs (20, 49, 50, 57, 87). RyR isoforms 1 and/or 2 appear essential for Ca2⫹ spark formation (20, 49), whereas RYR3 (57) or a spliced variant of this isoform (50) may be inhibitory to Ca2⫹ sparks. Preliminary studies in mouse cremaster feed arteries and their downstream arterioles are consistent with our functional studies performed in hamster vessels and indicate differences in expression of RyR isoforms that could underlie the functional differences between arteries and arterioles (55). Additional research will be required to determine whether differences in RyR isoform expression underlie the differences observed between feed arteries and arterioles. We found that smooth muscle cells in pressurized small arteries and their downstream arterioles, which develop similar levels of myogenic tone, consistently displayed Ca2⫹ waves in the absence of added agonist. Our observations are different AJP-Heart Circ Physiol • VOL

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from those obtained in rat small mesenteric arteries (64), where only 7% of cells generated Ca2⫹ waves due to pressureinduced myogenic tone, and recent studies from rat mesenteric and vas deferens arterioles and arteries, where Ca2⫹ oscillations were not observed in unpressurized vessels in the absence of vasoconstrictor agonists (10). These data support the notion that there are significant regional differences in mechanisms regulating and underlying Ca2⫹ signaling and myogenic tone. We found that a PLC inhibitor (U-73122, 10 ␮M) and two structurally different IP3R antagonists (xestospongin D, 5 ␮M; or 2-APB, 100 ␮M) nearly abolished the occurrence of Ca2⫹ waves, reduced global intracellular Ca2⫹, and inhibited myogenic tone in feed arteries and cremaster arterioles. These data support a large body of evidence implicating PLC in the genesis of myogenic tone (2, 17, 39, 46, 47, 69) and extend a recent report in vas deferens arterioles, suggesting a central role for IP3Rs in the regulation of agonist-induced arteriolar tone (10). Recent studies suggest that U-73122 may act to deplete intracellular Ca2⫹ stores (60). We do not think that the

Fig. 12. IP3 receptors and phospholipase C contribute to Ca2⫹ waves and myogenic tone in cremaster arterioles. Data are means ⫾ 95% confidence intervals for occurrence of Ca2⫹ waves (A, D, and G). All other data are means ⫾ SE for amplitude (F/Fo) and frequency (in Hz) of Ca2⫹ waves (A, D, and G); global fluo-4 intensity, an index of global intracellular Ca2⫹ (B, E, and H); and diameter (C, F, and I) in the absence (PSS) or presence of the IP3 receptor antagonists xestospongin D (5 ␮M, n ⫽ 5; A–C) or 2-APB (100 ␮M, n ⫽ 6; D–F); or the phospholipase C antagonist U-73122 (10 ␮M, n ⫽ 4 – 8; G–I). *P ⬍ 0.05, significantly different from value in PSS. Dashed lines in C, F, and I represent the maximum diameters of the vessels in 0 Ca2⫹ PSS. 300 • MAY 2011 •

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depletion of Ca2⫹ stores can explain the inhibitory effect of U-73122 on Ca2⫹ waves that we observed, because this PLC inhibitor had no effect on RyR-based Ca2⫹ sparks in feed arteries. In smooth muscle, RyRs and IP3Rs appear to access the same pool of Ca2⫹ (62). Thus the lack of effect of U-73122 on Ca2⫹ sparks argues against this agent working by depletion of intracellular Ca2⫹ stores in our studies. Furthermore, we found that the SERCA inhibitor thapsigargin (100 nM), while abolishing Ca2⫹ sparks and Ca2⫹ waves, also produced a significant increase in global Ca2⫹ and vasoconstriction in both feed arteries and arterioles, opposite the effects of U-73122 and the IP3R antagonists. Along with the lack of effect of U-73122 on Ca2⫹ sparks, this argues against store depletion as the primary means by which U-73122 produced effects in feed arteries and arterioles. Both 2-APB (9) and xestospongins (70) are known to have effects on other ion channels in addition to IP3Rs. As Ca2⫹ sparks in feed arteries were unaffected by these agents, we can rule out the depletion of intracellular stores or effects on RyRs as common off-target mechanisms. Thus we speculate that the fall in intracellular Ca2⫹ and consequent vasodilation observed with U-73122, xestospongin D, and 2-APB result from the inhibition of IP3R-based Ca2⫹ waves and the loss of Ca2⫹ signal amplification as a consequence of inhibited IP3 formation (U-73122) or blockade of IP3Rs (xestospongin D or 2-APB) and not because of possible off-target effects of the inhibitors. The inhibition of SERCA with thapsigargin (100 nM) also abolishes Ca2⫹ waves but results from the depletion of Ca2⫹ stores, the stimulation of Ca2⫹ entry through store-dependent mechanisms, and the likely depolarization-induced activation of Ca2⫹ influx through voltagegated Ca2⫹ channels as has been previously observed (59). Thus thapsigargin produced an increase in global Ca2⫹ and vasoconstriction in addition to the inhibition of Ca2⫹ sparks in feed arteries and Ca2⫹ waves in both feed arteries and arterioles. Calcium influx through voltage-gated Ca2⫹ channels and other ion channels importantly contributes to myogenic tone under normal conditions (23, 35). Our finding of a major role for the PLC-IP3-IP3R pathway in the regulation of global Ca2⫹ and myogenic tone suggests that IP3Rs may play a positive feedback role, likely amplifying Ca2⫹ signals from other sources and contributing to the Ca2⫹-dependent component of myogenic tone in small arteries and arterioles. In small arteries, while RyRs may contribute to this amplification (as we found that ryanodine inhibited not only Ca2⫹ sparks but also Ca2⫹ waves in hamster feed arteries), the major function of RyRs in the feed arteries appears to be in the negative feedback regulation of myogenic tone, as discussed above. Thus RyRs and IP3Rs serve distinct, contrasting roles in the regulation of myogenic tone in feed arteries, whereas only the positive feedback function of IP3Rs appeared functional in cremaster arterioles. We cannot exclude a possible modulatory role of the endothelium in our study, because all of the vessels used were endothelium intact. We do not think that any of the responses observed were strictly endothelium dependent for the following reasons. We have previously shown that endothelial cells from hamster second-order cremaster arterioles do not appear to express functional RyR, as caffeine (10 mM) does not elicit Ca2⫹ transients in these cells (18). Thus, at least in the arterioles, we do not think that the lack of effect of ryanodine AJP-Heart Circ Physiol • VOL

and tetracaine can be explained by some effect on endothelial cells. The inhibitors of the IP3 pathway (xestospongin D, 2-APB, and U-73122) all should inhibit endothelial cell Ca2⫹ signaling (18) and, if anything, tend to produce vasoconstriction. This is the opposite of what was observed in the feed arteries and arterioles. Conversely, thapsigargin should increase endothelial cell Ca2⫹ and produce vasodilation (63), again opposite of what was observed in both classes of vessel. However, we cannot exclude the possibility that the endothelium may have blunted some of the effects observed. This is a limitation of our experimental design. Overall, our studies demonstrate that there are important differences in the regulation of both smooth muscle cell Ca2⫹ signals and myogenic tone between feed arteries and their downstream arterioles. The well-documented negative-feedback role played by RyRs in the regulation of myogenic tone does not appear to hold true in second order hamster cremaster arterioles. In contrast, PLC and IP3Rs appear to function similarly in cremaster feed artery and arteriolar smooth muscle cells. The differences we observed between feed arteries and downstream arterioles caution the extrapolation of data gathered in arteries to arterioles and vice versa, even in closely related vasculatures. These differences in mechanisms also point to potential, novel therapeutic targets to modulate the tone of arterioles, for example, independent from upstream resistance arteries. Such microvessel-specific therapeutic targeting would allow the modulation of tissue blood flow and within-organ blood flow distribution potentially without substantial effects on systemic blood pressure and side effects, like orthostatic hypotension, which often result from drugs that nonspecifically target both resistance arteries and arterioles. ACKNOWLEDGMENTS We thank Dr. Steven S. Segal (University of Missouri) for input and suggestions and Erica L. Goodwin for outstanding technical assistance. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-32469 and PO1-HL-070687 and American Heart Association Fellowship 0815778G. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Abramoff MD, Magelhaes PJ, Ram SJ. Image Processing with ImageJ. Biophotonics International 11: 36 –42, 2004. 2. Bakker EN, Kerkhof CJ, Sipkema P. Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle. Cardiovasc Res 41: 229 –236, 1999. 3. Balasubramanian L, Ahmed A, Lo CM, Sham JS, Yip KP. Integrinmediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks. Am J Physiol Regul Integr Comp Physiol 293: R1586 –R1594, 2007. 5. Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart JO, Eble S, Klugbauer N, Reisinger E, Bischofberger J, Oliver D, Knaus HG, Schulte U, Fakler B. BKCa-Cav channel complexes mediate rapid and localized Ca2⫹-activated K⫹ signaling. Science 314: 615–620, 2006. 6. Berridge MJ. Smooth muscle cell calcium activation mechanisms. J Physiol 586: 5047–5061, 2008. 7. Boerman E, Jackson W. Ca2⫹-activated K⫹ channels are controlled by Ca2⫹ influx through voltage-gated Ca2⫹ channels, not the release of Ca2⫹ through ryanodine receptors in arteriolar smooth muscle (Abstract). FASEB J 22: 1142, 2008. 300 • MAY 2011 •

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