In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca2+ sparks activate large-conductance Ca2+-activated K+ (BK) channels leading to lowered SMC [Ca2+]i and vasodilation. Here we investigated whether Ca2+ sparks regulate SMC global [Ca2+]i and diameter in the spiral modiolar artery (SMA) by activating BK channels.
Methods
SMAs were isolated from adult female gerbils, loaded with the Ca2+-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca2+ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca2+ signals and vascular diameter were analyzed.
Results
Ca2+ sparks and waves were observed in pressurized SMAs. Inhibition of Ca2+ sparks with ryanodine increased global Ca2+ and constricted SMA at 40 cmH2O but inhibition of Ca2+ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH2O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH2O was abolished at 60 cmH2O, consistent with a greater Ca2+-sensitivity of constriction at 40 cmH2O than at 60 cmH2O. When the Ca2+-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH2O.
Conclusions
The results suggest that Ca2+ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca2+-sensitivity of constriction.
Cochlear function is sensitive to dynamic changes in cochlear blood flow that is responsible for the delivery of oxygen and glucose and the removal of CO2 [1, 2]. Regulation of cochlear blood flow is essential for hearing and is important as a treatment strategy for the restoration of hearing loss in humans [3–8]. Homeostatic regulation of blood flow in the cochlear capillary beds is achieved by the dynamic adjustment of the vascular diameter or “tone” of pre-capillary arteries and arterioles against systemic changes of pressure, nerve and metabolic activity [9–14]. The mechanisms involved in such regulation of the spiral modiolar artery, the principal artery of the cochlear blood supply, remain to be elucidated.
Smooth muscle cells of most arteries exhibit “Ca2+ sparks”, which are transient local elevations of Ca2+ caused by the opening of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) [15, 16]. In most smooth muscle cells, Ca2+ sparks activate BK channels, leading to membrane hyperpolarization, reduced activity of l-type voltage-dependent Ca2+ channels (VDCCs), decrease in [Ca2+]i and smooth muscle relaxation [15, 17–19]. Thus, the triad of Ca2+ sparks, VDCCs and BK channels effectively regulates intracellular Ca2+ to oppose vasoconstriction and maintain blood flow to the underlying tissue. Activation of BK channels by Ca2+ sparks is a potent vasodilatory mechanism to regulate SMC global [Ca2+]i and vascular diameter and a prominent feature in blood vessels of the cerebral, kidney, mesenteric and cardiac microcirculation [17–21].
We have recently demonstrated Ca2+ sparks in smooth muscle cells of the intact SMA [22]. In this study, we investigate whether Ca2+ sparks regulate the global Ca2+ and vascular diameter in the SMA. Our results demonstrate that Ca2+ sparks are also present in the pressurized SMA but do not regulate vasodilation of the SMA by activating BK channels. Instead, the effects produced by ryanodine, which eliminates Ca2+ sparks, are dictated by the pressure-dependent changes in the Ca2+ sensitivity of contraction.
Methods
Ethics statement
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Kansas State University (IACUC#: 2961 and 3245).
Isolation of the spiral modiolar artery (SMA)
Female gerbils between the ages of 4 to 12 weeks (Charles River, Wilmington, MA) were anesthetized with tri-bromo-ethanol (560 mg/kg i.p.) and sacrificed by decapitation. Auditory bullae were harvested and the spiral modiolar arteries (SMAs) separated from the cochlea by microdissection in HEPES-buffered physiological saline solution (PSS) at 4 C°.
Pressurization and superfusion
Segments of the SMA were pressurized and perfused in a custom-built bath chamber using a variable hydrostatic pressure column connected to a motorized set of concentric glass pipettes (Wangemann Instruments, Kansas State University, KS) mounted on an inverted microscope (Axiovert 200, Carl Zeiss, Göttingen, Germany) [12]. Briefly, arteries were held by a holding pipette and luminally perfused with a perfusion pipette at one end while the other end was occluded using a blunt glass pipette. All pipettes were prepared using a custom-built micro-forge. The pressurized vessel was superfused in the bath with either HEPES-buffered PSS at a rate of 1.6 ml/min, permitting one complete exchange of the bath volume (~70 μl) within ~3 s. Experiments were conducted at 37 °C. Bath temperature was maintained by a triple heating system consisting of regulating the temperatures of the superfusate (8-line heater, CL-100, Warner Instruments, Hamden, CT, USA), the bath chamber (TC 324B, Warner Instruments) and the microscope objective (TC 324B, Warner Instruments).
Measurements of cytosolic Ca2+ signals
Cytosolic Ca2+ signals in SMCs were monitored as spatial and temporal changes in the fluorescence intensity of the indicator dye fluo4. For loading the dye, pressurized vessel segments were incubated in 2.5 μM fluo4-AM (Invitrogen, Carlsbad, CA, USA) for 15 min at 37 °C, followed by wash and superfusion with HEPES-buffered PSS. The dye loaded virtually exclusively into SMCs. Fluo4 was excited by a 488 nm argon laser. Fluorescence emissions were filtered by a 488 notch and two long-pass filters (490 nm and 505 nm) and recorded by a photomultiplier through an open pinhole (LSM 510 Meta, Carl Zeiss).
Ca2+ sparks
Ca2+ sparks in SMCs of pressurized SMA were detected in frame scans and line scans. For frame scans, tangential images of the vascular wall (32.14 μm × 10.04 μm) were recorded using an oil-immersion objective (Plan-Neofluar 40× 1.3 N.A., Carl Zeiss) at a temporal resolution of ~61 images/s (16.3 ms/frame) and a spatial resolution of 0.25 μm × 0.25 μm per pixel. Spark sites were identified using custom-designed software, SparkAn, developed by Dr. Adrian D. Bonev (University of Vermont, VT, USA) in IDL 5.0.2 (Research Systems, Boulder, CO) and kindly provided for use by Dr. Adrian D. Bonev and Dr. Mark T. Nelson (University of Vermont). Ca2+ sparks were detected by dividing an area spanning 2.01 μm (8 pixels) × 2.01 μm (8 pixels) in each frame by a baseline (F0) that was obtained by averaging 10 frames without Ca2+ spark activity. Spark traces and 2-dimensional pseudo-color images were obtained as F/F0. 3-dimensional ratio images were generated by SparkAn.
Line-scan recordings of 5 s duration each were performed at a Ca2+ spark site to determine the temporal parameters of Ca2+ sparks in SMCs. Lines (0.15 μm × 12.4 μm) were recorded using an oil-immersion objective (Plan-Neofluar 40× 1.3 N.A.) at a temporal resolution of ~521 lines per second (0.82 ms per line). For spark measurements at different pressures, three 5 s line-scans were performed first at 60 cmH2O, followed by three line-scans at 40 cmH2O, followed by three line scans at 60 cmH2O. Time intervals between consecutive line-scans were 15 s to allow for recovery. Time intervals between pressure-changes were 45 s. For experiments in ryanodine and tetracaine, 10 μM ryanodine or 100 μM tetracaine was introduced after the third line-scan in PSS and scans were resumed after 2 min. Recordings were analyzed as described earlier [11]. For presentation, a single 5 s line-scan image was contrast-enhanced to highlight the occurrence of Ca2+ spark events.
Determination of length and height of smooth muscle cells
To calculate Ca2+ spark density, cell length and cell height of single SMCs were estimated from scanned images of pressurized SMA loaded with BCECF (Sigma-Aldrich). SMC length and height were determined to be 132 ± 17 μm and 3.2 ± 0.1 μm based on images of 9 vessels that each covered 20 – 30 cells. These values correspond to a cell volume of ~1 pl, which is consistent with SMCs from other vessels [15].
Simultaneous measurements of vascular diameter and global cytosolic Ca2+
Pressurized vessels (40 or 60 cmH2O), loaded with the indicator dye fluo4 as described above, were superfused with HEPES-buffered PSS. To record the inner diameter of pressurized vessels simultaneously with changes in the global cytosolic Ca2+ in SMCs, images (225 μm × 225 μm) were recorded using an oil-immersion objective (Plan-Neofluar 40× 1.3 N.A.) with a temporal resolution of 1 image/s (983 ms/frame) and a spatial resolution of 0.44 μm × 0.44 μm per pixel. In addition to filtering and recording fluorescence emissions as described above, the transmitted light from the argon laser was detected by a second photomultiplier. Inner diameter was measured by automatic edge detection by a method developed and described in Reimann et al. 2011 [12]. Inner diameter (ID) was detected from acquired real time transmitted light images, using a custom written data acquisition program (Dr. W. Gil Wier, University of Maryland). Edge detection data were analyzed using a custom written analysis program in Origin 6.0 (Dr. P. Wangemann, Kansas State University). Inner diameter changes were normalized against the average of 30 data points obtained in PSS at the beginning of the experiment (basal vascular tone). Fluorescence intensity values from 5–10 SMCs per pressurized vessel were averaged and normalized between the fluorescence values in Ca2+ free solution and the fluorescence value in PSS before the addition of drugs according to the formula Norm Ca2+ = (([Ca2+]i ‐ [Ca2+]0)/([Ca2+]PSS ‐ [Ca2+]0)) + 1, where [Ca2+]0 is marked b and [Ca2+]PSS is marked in Figs. 2b, 3b, 3e, 4b, 5b, and 7b. a
Ca2+ sensitivity
Simultaneous measurements of diameter and global cytosolic Ca2+ were performed to determine the Ca2+-sensitivity of constrictions. In these experiments, following equilibration in HEPES-buffered PSS for 15 min, arteries were superfused with saline solutions containing 0, 1, 3 and 10 mM Ca2+ in 2 min steps, first at 60 cmH2O followed by the same protocol at 40 cmH2O. Data points for concentration curves were obtained by averaging diameter and fluorescence intensity measurements over the last 30s of each Ca2+ step and normalizing against the average value obtained in PSS at 60 cmH2O. Data points from individual vessels were fitted to a modified Hill equation:
where Dia is the normalized diameter, Max is the diameter at 60 cmH2O in PSS containing 1 mM Ca2+, Base is the maximum achievable constriction with respect to Max for the female gerbil SMA estimated from previous observations [23], [Ca2+] is the normalized cytosolic Ca2+ concentration, h is the slope coefficient, and FC50 is the fold-change in the global cytosolic Ca2+ concentration that is necessary for a half-maximal constriction. The slope coefficient h was set to -5.2 and Max was clamped to 100%. Two FC50 values, one each for 60 cmH2O and 40 cmH2O, were obtained for each experiment. For presentation, normalized Ca2+ and diameter data were averaged and fitted with the equation above using average FC50 values.
Solutions and drugs
HEPES-buffered PSS contained (in mM): 150 NaCl, 5 HEPES, 3.6 KCl, 1 MgCl2, 1 CaCl2 and 5 glucose, pH adjusted to 7.4 at 37 °C. Ca2+-free solutions were devoid of CaCl2 and contained 1 mM EGTA. 100 μM papaverine hydrochloride (Pap) was added to the Ca2+ free solution wherever indicated. Stock solutions of ryanodine (Ryn, 20 mM, Enzo Life Sciences, NY or Santa Cruz Biotechnologies, Santa Cruz, CA), tetracaine (Tet, 100 mM, Sigma- Aldrich), paxilline (Pax, 10 mM, Sigma-Aldrich) and papaverine (Pap, 250 mM, Sigma-Aldrich) were prepared in DMSO and stored at -20 °C and freshly diluted to target concentration in solution when required taking care that the final DMSO concentration in solution did not exceed 0.1%. Endothelin-1 (ET-1, 1 μM, Sigma-Aldrich) and iberiotoxin (IbTx, 2 μM, Alomone Labs, Jerusalem, Israel) were always freshly prepared in PSS for immediate use.
Results
Ca2+ sparks in the pressurized spiral modiolar artery
Ca2+ sparks and waves were recently reported in the intact unpressurized gerbil SMA [22]. We now report Ca2+ sparks and Ca2+ waves in SMCs of the pressurized gerbil SMA (Fig. 1). Ca2+ spark sites were consistently observed in frame scans (Fig. 1a), with super Ca2+ sparks, of larger cross-sectional area and longer duration of elevated Ca2+ observed occasionally, which may reflect the combined synchronized activity of two or more closely spaced Ca2+ sparks sites. The average spatial area of spark sites was 14 ± 3 μm2, corresponding to a spatial width of ~4 μm. Two out of 13 recorded Ca2+ spark sites had larger spatial areas between 25 – 50 μm2 (Fig. 1b). An average of 1.3 ± 0.2 Ca2+ spark sites were present in 200 ± 10 μm2 of recorded area (n = 10), corresponding to ~47% of one cell, giving a spark density of 2.8 ± 0.3 spark sites/cell. The frequency of Ca2+ spark occurrence per site and other temporal parameters were measured in pressurized SMA using line-scans (Fig. 1c). Spark frequency per site increased from 0.6 Hz to 0.9 Hz, as pressure increased from 40 cmH2O to 60 cmH2O (Table 1). However, increasing pressure from 40 to 60 cmH2O did not alter the spark amplitude, the rise-time or the half-time of decay (Table 1). Ca2+ sparks were completely eliminated in the presence of 10 μM ryanodine (Fig. 1c) and significantly decreased in frequency in the presence of 100 μM tetracaine (Fig. 1d). Ca2+ oscillations exhibiting wave-like phenomena were also observed in SMCs and were likewise abolished by application of 10 μM ryanodine (Fig. 2a).
Fig. 1
Ca2+ sparks in smooth muscle cells of the pressurized spiral modiolar artery. a Ca2+ spark site captured during a 5 s frame scan of SMCs in a pressurized (60 cmH2O) SMA. Grey panel (top panels) depicts the average of 10 frames that do not contain a Ca2+ spark. Outline (in red) depicts the visible portion of the cell. Scale bar = 2 μm. Pseudo-color image depicts an average (left middle panel) and a large (right middle panel) Ca2+ spark at its peak. Regions of interest (ROIs) are 2 μm × 2 μm. (Bottom panels) show a 3D rendering of the 2D images. Traces (green and red) represent the fluorescence intensity changes occurring at the corresponding ROIs shown in the panels above. b Histogram showing the distribution of the calculated spatial area of Ca2+ spark sites. c Contrast-adjusted 5 s line-scan recording of a Ca2+ spark site in a SMC of a pressurized (60 cmH2O) SMA and the corresponding fluorescence intensity traces recorded in PSS and in the presence of 10 μM ryanodine (Ryn). d Spark frequency in PSS (n = 9), 100 μM tetracaine (Tet, n = 3) and 10 μM ryanodine (Ryn, n = 4)
Table 1
Parameters of Ca2+ sparks
Pressure (cmH2O)
Frequency per site (Hz)
Amplitude (F/F0)
Rise-Time (ms)
Half-decay-time (ms)
40
0.6 ± 0.1 (n = 17)
1.53 ± 0.03 (n = 65)
18.3 ± 0.7 (n = 65)
19.8 ± 1.2 (n = 64)
60
a 0.9 ± 0.1 (n = 17)
1.49 ± 0.03 (n = 110)
16.9 ± 0.3 (n = 110)
17.8 ± 0.8 (n = 110)
a indicates significance between parameter values measured at 40 and 60 cmH2O
Fig. 2
Inhibition of Ca2+ sparks with ryanodine increases the global [Ca2+]i and constricts the SMA. a Representative recordings of [Ca2+]i changes from single smooth muscle cells from a pressurized (40 cmH2O) SMA in response to 10 μM ryanodine (Ryn). b Average of normalized traces of [Ca2+]i changes at 40 cmH2O (48 cells). Traces in a and b were normalized as described in Methods. c Average trace of corresponding changes in vascular diameter (6 arteries). Diameter changes were normalized to the average of values recorded between 30–60 s (average value indicated by ‘a’ was set to 1). [Ca2+]i and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10 s
Effects of inhibitors of Ca2+ sparks and BK channels on global Ca2+ and vascular diameter of the SMA
In most arteries, inhibition of Ca2+ sparks and/or BK channels has been shown to increase SMC global Ca2+ to cause a robust vasoconstriction in a non-additive fashion, reflecting that Ca2+ sparks and BK channels are part of the same mechanism to hyperpolarize the membrane and limit Ca2+ influx, leading to vasorelaxation [17, 18, 20]. In the pressurized (40 cmH2O) SMA, application of 10 μM ryanodine inhibited Ca2+ sparks and appeared to similarly increase the average global cytosolic Ca2+ followed by a robust vasoconstriction (Fig. 2). However, application of 100 μM tetracaine, another known inhibitor of ryanodine receptors and Ca2+ sparks [24], or application of 100 nM iberiotoxin, a potent BK channel inhibitor, did not cause any change in global Ca2+ or vascular diameter similar to that produced by ryanodine (Fig. 3). Activation of BK channels is dependent on the local [Ca2+]i as well as the membrane potential of the smooth muscle membrane [25]. It is possible that lack of an effect of iberiotoxin is a consequence of unopened BK channels caused by a hyperpolarized resting membrane potential in the smooth muscle cells of the SMA. To account for such a possibility, the pressurized SMA was superfused with PSS solution containing 30 mM K+. High K+ induced a transient increase in the global [Ca2+]i and vasoconstriction. Under these conditions, 100 nM iberiotoxin remained without effect (Fig. 4). Furthermore, contrary to the effect at 40 cmH2O, 10 μM ryanodine increased global Ca2+ modestly and did not constrict the SMA pressurized at 60 cmH2O (Fig. 5), even though spark frequency is increased significantly from 40 to 60 cmH2O (Table 1).
Fig. 3
Inhibition of BK channels with iberiotoxin or inhibition of Ca2+ sparks with tetracaine does not increase the global [Ca2+]i or constrict the SMA at 40 cmH2O. a Representative recordings of [Ca2+]i changes from single smooth muscle cells from a pressurized (40 cmH2O) SMA in response to 100 nM Ibtx. b Average of normalized traces of [Ca2+]i changes in the presence of Ibtx (65 cells). Traces in a and b were normalized as described in Methods. c Average trace of corresponding changes in vascular diameter in the presence of Ibtx (8 arteries). d Representative recordings of [Ca2+]i changes from single smooth muscle cells from a pressurized (40 cmH2O) SMA in response to 100 μM Tet and 1 μM nifedipine (Nif). e Average of normalized traces of [Ca2+]i changes at 40 cmH2O (26 cells). Traces in d and e were normalized as described in Methods. f Average trace of corresponding changes in vascular diameter (5 arteries) in the presence of Tet and Nif. Diameter changes were normalized to the average of values recorded between 30–60s (value indicated by ‘a’ was set to 1). [Ca2+]i and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10s
Fig. 4
Inhibition of BK channels in the presence of high K+ does not increase the global [Ca2+]i or constrict the SMA. a Representative recordings of [Ca2+]i changes from single smooth muscle cells from a pressurized (40 cmH2O) SMA in the presence of PSS containing 30 mM K+ and 100 nM Ibtx. b Average of normalized traces of [Ca2+]i changes in the presence of PSS containing 30 mM K+ and Ibtx (36 cells). Traces in a and b were normalized as described in Methods. c Average trace of corresponding changes in vascular diameter (6 arteries). Diameter changes were normalized to the average of values recorded between 30 – 60 s (value indicated by ‘a’ was set to 1). [Ca2+]i and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10 s
Fig. 5
Inhibition of Ca2+ sparks with ryanodine results in a modest increase in global [Ca2+]i but does not constrict the SMA at 60 cmH2O. a Representative recordings of [Ca2+]i changes from single smooth muscle cells from a pressurized (60 cmH2O) SMA superfused with HEPES-buffered PSS in response to 10 μM Ryn. b Average of normalized traces of [Ca2+]i changes at 60 cmH2O (54 cells). Traces in a and b were normalized as described in Methods. c Average trace of corresponding changes in vascular diameter (6 arteries). Diameter changes were normalized to the average of values recorded between 30 – 60 s (average value indicated by ‘a’ was set to 1). [Ca2+]i and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10 s
These effects suggest that, unlike cerebral arteries, the ryanodine-induced increase in global Ca2+ and constriction at 40 cmH2O in the SMA may not be attributed to a loss of the hyperpolarizing influence of the Ca2+ spark-BK channel signaling mechanism. Under these conditions, ryanodine-sensitive Ca2+ sparks do not appear to regulate global Ca2+ and vascular tone via BK channels in the SMA. This raises the question as to the mechanism involved in the ryanodine-induced increase in the global Ca2+ and vasoconstriction. It is to be expected that at least a portion of the global Ca2+ is the result of Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs), which are open at the physiological resting membrane potential in SMCs. Evidence for active VDCCs in the SMA comes from the observation of a decrease in global Ca2+ upon application of a reversible VDCC inhibitor, 2 μM nifedipine, in the presence of 100 μM tetracaine (Fig. 3d and e). It is possible that the remainder of the ryanodine-induced increase in the global Ca2+ is the result of ryanodine receptor-mediated Ca2+ release from the sarcoplasmic reticulum (SR). It is well-established that at a concentration of 10 μM, ryanodine binds to open ryanodine receptors and modifies the channel to lock them in an irreversible sub-conductance state of 234 pS [16, 26] that inhibits Ca2+ release and instead “leaks” SR Ca2+ into the cytosol. This is reflected in the transient increase in global Ca2+ immediately upon application of 10 μM ryanodine, followed by a slowly decaying plateau phase devoid of Ca2+ oscillations, indicating the relatively slow emptying of the SR through the partially open ryanodine receptors (Fig. 2a and b).
It is to be noted that the ryanodine-induced vasoconstriction continues to increase as the corresponding average global Ca2+ plateaus and then decreases (Fig. 2b and c). Indeed, the maximum vasoconstriction corresponds to the least increase in global Ca2+ induced by ryanodine, suggesting an increase in the Ca2+ sensitivity of constriction following the initial increase in the global Ca2+. In other words, the constriction induced by ryanodine at 40 cmH2O may be attributed to enhanced Ca2+ sensitivity of the SMA that is able to respond to the ryanodine-induced increase in intracellular Ca2+ with vasoconstriction. The observation that the ryanodine-induced constriction at 40 cmH2O is enhanced (Fig. 2c) compared to that at 60 cmH2O (Fig. 5c) suggests that the Ca2+ sensitivity at 40 cmH2O may be greater than at 60 cmH2O.
Ca2+ sensitivity of the SMA decreases with increasing pressure
The Ca2+ sensitivity at 40 and 60 cmH2O was determined from simultaneous measurements of the cytosolic Ca2+ and the vascular diameter. The cytosolic Ca2+ concentration was manipulated by altering the Ca2+ concentration in the superfusate (Fig. 6a and b). Normalized cytosolic Ca2+ and corresponding vascular diameter measurements were plotted against each other and fitted to the Hill equation. A decrease in the pressure from 60 to 40 cmH2O shifted the Ca2+-diameter relationship to the left on the Ca2+ axis, indicating a dramatic increase in the Ca2+ sensitivity, with nearly 2-fold decrease in the Ca2+ required for a half-maximal constriction at 40 cmH2O compared to that at 60 cmH2O (Fig. 6c), whereas a time control repeated at 60 cmH2O did not (Fig. 6d). Thus, the modest increase in global Ca2+ caused by ryanodine at 60 cmH2O was insufficient to constrict the SMA at this pressure, whereas the enhanced Ca2+ sensitivity at 40 cmH2O allowed for a robust constriction for an increase in global Ca2+.
Fig. 6
Ca2+ sensitivity of the SMA decreases with increase in pressure. a, b Vascular diameter and [Ca2+]i changes in the presence of 0, 1, 3 and 10 mM Ca2+ were simultaneously measured from vessel segments pressurized at 60 cmH2O (black trace) followed by 40 cmH2O (red trace), as indicated. a Summary of changes in [Ca2+]i measured as changes in fluorescence intensity. b Summary of corresponding diameter measurements first at 60 cmH2O and then at 40 cmH2O (4 arteries). Data were acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10s. c Ca2+ sensitivity of SMA at 60 cmH2O (black trace) and 40 cmH2O (red trace). d Ca2+ sensitivity of SMA at 60 cmH2O (black trace) and time control at 60 cmH2O (grey trace). Numbers next to symbols represent the number of arteries. For c and d, average FC50 values are given as mean ± sem. Data points for normalized [Ca2+]i and diameter were obtained by averaging diameter and fluorescence intensity measurements over the last 30 s of each Ca2+ step and normalizing these values against the average value obtained in PSS containing 1 mM Ca2+ at 60 cmH2O (denoted as ‘a’)
Endothelin enhances the ryanodine-induced vasoconstriction
The result above implies that conditions that increase the Ca2+ sensitivity at 60 cmH2O would increase the ryanodine-induced vasoconstriction. Consequently, the SMA pressurized at 60 cmH2O was first exposed to 1 nM endothelin-1 (ET-1) for 1 min. It has been previously shown that endothelin-1 acts via ETA receptors to increase the Ca2+ sensitivity of the SMA in a rho-kinase dependent manner [27]. ET-1 caused a transient increase in the cytosolic Ca2+ concentration and a persistent vasoconstriction, consistent with an increase in the Ca2+ sensitivity (Fig. 7). Under these conditions, 10 μM ryanodine caused a vasoconstriction that was enhanced compared to that observed in the absence of ET-1 (Fig. 5). These results support the concept that ryanodine increases global Ca2+ and constricts the SMA when the Ca2+ sensitivity is high. Ca2+ sensitivity of SMC contraction is hence a critical factor in the regulation of the vascular diameter of the SMA in response to changes in pressure and cytosolic global Ca2+.
Fig. 7
Inhibition of Ca2+ sparks with ryanodine increases global Ca2+ and constricts SMA at 60 cmH2O following an increase in Ca2+ sensitivity by endothelin-1. a Representative recordings of cytosolic Ca2+ changes from single smooth muscle cells from a SMA loaded with fluorescent dye fluo4 and pressurized to 60 cmH2O in response to 10 μM ryanodine after treatment with 1 nM endothelin-1 (ET-1). b Average of normalized traces of cytosolic Ca2+ changes at 60 cmH2O (64 cells from 7 arteries). Traces in a and b were normalized as described in Methods. c Average trace of corresponding changes in vascular diameter of SMA pressurized at 60 cmH2O (15 arteries). Diameter changes were normalized to the average of values recorded between 30–60 s (value indicated by ‘a’ was set to 1). Ca2+ and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10 s
Discussion
Salient findings of the present study are 1) Ca2+ spark frequency in the pressurized SMA increases with pressure; 2) Inhibition of Ca2+ sparks with ryanodine increases global Ca2+ and causes a robust vasoconstriction, however, ryanodine-induced effects on global Ca2+ and vascular diameter are not reproduced by other inhibitors of Ca2+ sparks or by inhibitors of BK channels as would be expected if Ca2+ sparks activated BK channels to regulate membrane potential, global Ca2+ and vascular diameter. 3) The ryanodine-induced vasoconstrictions depends on the Ca2+ sensitivity, which is higher at 40 cmH2O compared to that at 60 cmH2O and can be enhanced with endothelin-1.
Ca2+ sparks
Ca2+ sparks in the pressurized SMA occurred with a lower frequency than in unpressurized SMA [22], but with a similar frequency, spatial width and spark site density as observed in smooth muscle cells of cerebral pial arteries [15, 19, 28] and pressurized mesenteric arteries [21, 29]. The increase in Ca2+ spark frequency in response to a 20 cmH2O (~ 14 mmHg) increase in pressure, is also consistent with observations made in cerebral arteries [28]. However, as in unpressurized SMA, the time of half decay of Ca2+ sparks was far shorter (~17–19 ms) than that observed for Ca2+ sparks in cerebral arteries, but closer to that found in rat heart [16, 30]. Ca2+ spark amplitudes and decay times are generally a reflection of the number as well as the isoform of ryanodine receptors (RyRs) present in a spark cluster. Typically, Ca2+ spark sites are composed of 4 – 6 RyRs, giving a punctate staining pattern in immunolocalization studies. In the SMA, the distribution pattern of RyRs in SMCs shows a uniform expression throughout the SR rather than a punctate expression expected of ryanodine receptors clustered in spark sites [22] and may underlie the observed differences in the temporal properties and functional role of Ca2+ sparks in the SMA.
Absence of the Ca2+ spark/BK channel hyperpolarizing mechanism in the SMA
The Ca2+ spark/BK channel signaling complex provides an important vasodilatory mechanism in preventing or mitigating pressure- or agonist-induced vasoconstrictions in arteries. This negative feedback mechanism in regulating vascular tone is evident from observations that pharmacological inhibition of Ca2+ sparks and/or BK channels or SMC-specific genetic manipulation of BK channel or RyR expression leads to the loss of this hyperpolarizing signal leading to membrane depolarization, increased VDCC activation, increase in Ca2+ influx and global Ca2+ and enhanced vasoconstriction [18, 20, 31–33]. However, Ca2+ sparks have not always been linked to a hyperpolarizing or vasodilatory mechanism. Other studies have observed excitatory roles for Ca2+ sparks and RyR-mediated Ca2+ release in small diameter arterioles. Kur et al. [24] reported that, contrary to the conventional hyperpolarizing mechanism, Ca2+ sparks in retinal arterioles combined to form Ca2+ waves and enhanced the myogenic tone. Westcott et al. [34] reported SMCs of murine cremaster muscle feed arterioles to express diffused staining of RyRs without manifesting Ca2+ sparks and no coupling with BK channels, while SMCs of upstream feed arteries exhibited clustered staining of RyRs, robust Ca2+ sparks and spatial and functional coupling to BK channels, indicating heterogeneity of RyR function within the same vascular tree. In the SMA, the effects of ryanodine on global Ca2+ and vascular diameter at 40 cmH2O seemed to suggest, at first, a vasodilatory mechanism for Ca2+ sparks acting via BK channels. However, the failure of tetracaine, an RyR inhibitor, which inhibits Ca2+ sparks without depleting the SR, and iberiotoxin, a BK channel inhibitor, to produce similar effects on global Ca2+ and diameter as ryanodine (Figs. 2 and 3) combined with the non-effect of BK channel inhibition following membrane depolarization by external application of high K+ (Fig. 4) or increase in pressure (Fig. 5) disproves the regulation of vascular tone of the SMA by the Ca2+ spark/BK channel mechanism.
Regulation of vascular tone by Ca2+ sensitivity
Changes in SMC global Ca2+ have generally been accepted as the central mechanism regulating SMC contractility in the development of pressure-dependent myogenic tone [35]. More recently, the contribution of Ca2+-independent processes that regulate the Ca2+ sensitivity of the myofilament in the development of myogenic tone have been better described [36]. In cerebral and skeletal resistance arteries, increases in intravascular pressure are associated with increases in the Ca2+ sensitivity achieved by balancing the relative activities of myosin light chain kinase and myosin light chain phosphatase in a PKC and rho-kinase-dependent manner. Such changes in Ca2+ sensitivity further augment the Ca2+-dependent myogenic vasoconstrictions [36–40]. We have previously shown that myogenic tone in the male, but not female, gerbil SMA is regulated not by changes in the SMC global Ca2+ but by rho-kinase-mediated changes in the Ca2+ sensitivity of contraction, which was revealed under inhibition of NO-mediated signaling [23]. The present study shows that the regulation of vascular tone in the female gerbil SMA is also determined by the Ca2+ sensitivity of the myofilament, with the crucial difference that increase in intravascular pressure significantly lowered the Ca2+ sensitivity (Fig. 7). This finding is consistent with the development of small myogenic tones with increasing intravascular pressures in the SMA [12]. Rho-kinase-dependent regulation of Ca2+ sensitivity also plays a significant role in mediating the vascular effects of endogenous vasoconstrictors and agonists [41–43]. Further studies are required to elucidate the mechanisms underlying the relationship between pressure and Ca2+sensitivity in the SMA.
Conclusions
In conclusion, in this study, we have shown in the gerbil spiral modiolar artery that Ca2+ sparks, while present, do not regulate vascular tone and global Ca2+ by activating BK channels. Instead, ryanodine-receptor mediated increases in global Ca2+ and vasoconstriction depend on the Ca2+ sensitivity of SMC contraction, which is enhanced at lower pressures or by regulating rho-kinase activity.
It remains to be seen whether such mechanisms of vascular tone regulation as described in this study are applicable to spiral modiolar arteries in general or particularly unique to the gerbil spiral modiolar artery. Gerbils are commonly favored over other rodents such as mice and rats as hearing models for investigations into the causes for age-related hearing loss involving pathological changes in both peripheral and central auditory system components. Gerbils are uniquely suited for such investigations as they exhibit sensitive hearing in the low frequency ranges (below 4 kHz) that are relevant for human auditory perception, compared to the much higher thresholds in mice and rats for the same frequency range [44]. Thus, the differences observed in the regulation of the gerbil SMA by Ca2+ sparks and BK channels compared to the observations made in arteries from other extensively studied rodent species become relevant in the choice of appropriate models for future interpretation of hearing studies and pharmacological interventions.
Declarations
Funding
This study was supported by NIH-R01-DC04280 to PW and by Fortüne 2339-0-0 (University of Tuebingen, Tuebingen, Germany) to KR. The Confocal Microscopy Core facility was supported by the College of Veterinary Medicine at Kansas State University and by NIH-P20-RR017686.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Authors’ contributions
GK and PW conceived and designed the study. GK collected and analyzed the data. KR collected and analyzed the data for Ca2+ sparks in ryanodine and Ca2+ sensitivity at different pressures. PW wrote the software code for data analysis. GK, KR and PW wrote the manuscript. All authors have read and agreed to the final version of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent to publication
Not applicable.
Ethics Approval
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Kansas State University (IACUC#: 2961 and 3245).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
(1)
Anatomy & Physiology Department, Cell Physiology Laboratory, Kansas State University
(2)
Department of Otolaryngology–Head and Neck Surgery, Tübingen Hearing Research Centre, and Molecular Physiology of Hearing, University of Tübingen
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