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,1
* The iCAPTUR4E Center, University of British Columbia, St. Paul’s Hospital, Vancouver, BC, and Cardiovascular Sciences, Children’s and Women’s Health Centre of British Columbia, Vancouver, BC, Canada; and
Pharmacology Group, School of Pharmacy, University of Lausanne, 1015 Lausanne, Switzerland
2Correspondence: Cardiovascular Research, BC Research Institute for Children’s and Women’s Health, 2082–950 W. 28th Ave., Vancouver BC, Canada V5Z 4H4. E-mail: breemen{at}interchange.ubc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: calcium • mitochondria • sarcoplasmic reticulum • SMC
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although mitochondrial Ca2+ transport has been studied for many years, its role in the shaping of spatio-temporal Ca2+ signaling patterns has not been thoroughly examined in vascular smooth muscle. The low-affinity, high-capacity Ca2+ uniporter, which is driven by the large electrical potential across the inner membrane (due to proton extrusion via the respiratory chain) (2)
and located in the inner mitochondrial membrane, has a Kd for Ca2+ of 10–20 µM and is minimally active in the submicromolar range (2
, 3)
. Therefore, it was previously thought that the MT would not sense [Ca2+]i under physiological conditions. However, with the discovery of various SR-related cytoplasmic microdomains such as in the superficial buffer barrier (4)
and transient localized Ca2+ sparks activating Ca2+-activated potassium channels (5)
it seemed likely that such microdomains for Ca2+ might be related to other organelles. This idea was in fact proven by the elegant experiments done by Pozzan and co-workers (for a review see, ref 6
) using targeted aequorin technology to specifically localize a Ca2+ sensing probe to the inner mitochondrial matrix of HeLa cells (7
, 8)
. Aequorin is exquisitely suited for measuring calcium concentrations in the range where MT are active as it is sensitive between 0.1 and 10 µM (9)
and can be accurately calibrated.
Mitochondria contain many Ca2+-sensitive enzymes, mainly dehydrogenases, such that uptake of Ca2+ by MT (via agonist stimulation) increases cellular ATP production in anticipation of cellular needs (10
, 11)
. It has been shown that these organelles are capable of taking up Ca2+ under physiological conditions if high concentrations of local Ca2+ are sensed by the uniporter during stimulation by a variety of inositol-1,4,5-trisphosphate (IP3)-generating agonists (12
, 13)
. These results have been validated by subsequent electron microscopy and GFP studies (14)
, which have revealed close appositions of the ER and MT membranes in HeLa cells.
In an earlier study we examined the role of the MT in regulating Ca2+ in a skeletal muscle cell line (15)
; however, few have looked specifically in vascular smooth muscle as it is difficult to transfect large plasmids, such as aequorin, in this tissue. However, with new methods for transfection, this technique can now be extended to cultured smooth muscle cells in order to directly examine the contribution that MT may make in regulating [Ca2+]i.
Using the above approach, we have examined mitochondrial Ca2+ signals in response to ATP stimulation of vascular smooth muscle cells in culture transiently transfected with a mitochondrially targeted aequorin. The data generated indicate that MT share cytoplasmic microdomains with the SR, which, during SR Ca2+ release, have local [Ca2+] 10-fold greater than the average [Ca2+]i. The plasma membrane Na+/Ca2+ exchanger (NCX) indirectly modulates the Ca2+ signals of half of the MT.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, grown up as a feeder culture, suspended in 90% DMEM, 10% DMSO, and stored in aliquots in liquid nitrogen. Cells were used between passages 6–11 and were grown in DMEM supplemented with 10% fetal calf serum, 100 units/mL penicillin G, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, MEM vitamin solution, and MEM amino acid solution (Life Technologies, Grand Island, NY). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
SMC phi cell line transiently expressing aequorin targeted to MT
SMCs were transiently transfected with a pcDNAI expression vector containing a cDNA-encoding aequorin targeted to the MT (13)
. Smooth muscle cells were seeded on Matrigel-coated ThermanoxTM coverslips of 13 mm diameter in 4-well plates (Nunc, Life Technologies). After 1 day in culture, cells in 4-well plates were transfected with the following solution: 4 µg of mtAeq/pcDNAI (1ug per well) in 12.5 µL of Effectene® reagent in serum-containing medium. Cells were used after 3 days in culture. Photon quantification after digitonin (100 µM) permeabilization showed that cells exhibited sufficient mtAeq expression for quantitative [Ca2+]m analysis.
Mitochondrial [Ca2+] measurement
SMCs were seeded on Matrigel-coated ThermanoxTM coverslips of 13 mm diameter (Nunc, Life Technologies, Inc.). After 3 days in culture, [Ca2+]m was measured in a population of smooth muscle cells (
60,000 cells/coverslip). The mtAeq was reconstituted with coelenterazine (5 µM) in DMEM for 2–4 h before the experiment. The coverslip was held in a 0.5 mL chamber heated constantly at 37°C and positioned 5 mm from the photon detector. Cells were superfused at a rate of 1 mL/min with physiological salt solution (PSS, in mM: NaCl 145, KCl 5, MgCl2 1, HEPES 5, glucose 10, and CaCl2 1.2, pH 7.4). Stimuli were usually applied for 5 min (unless state otherwise) in PSS. Emitted luminescence was detected by a photomultiplier apparatus (EMI 9789, Thorn-EMI, UK) and recorded every second using a computer photon counting board (EMI C660) as described previously (17)
As published (18
19
20
21)
, the relationship between recorded counts and [Ca2+] is shown in Equation 1
,
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Cytosolic [Ca2+] measurement
SMCs were seeded on glass coverslips of 22 mm diameter. After 40 min loading in 5 µM Fura-2 AM in the dark at room temperature, cells were washed six times with PSS. The coverslip was placed in a thermostated chamber at 37°C on the stage of a fluorescence microscope (Nikon Diaphot, Küsnacht, Switzerland). After 3 min of stabilization in PSS, smooth muscle cells were excited at alternative wavelengths of 340 and 380 nm and emission was recorded at 510 nm. The PhoCal software (Life Science Resources Ltd., Cambridge, UK) was used to analyze the collected data. The ratio R between the emitted light at 340 and 380 nm permitted calculation of [Ca2+]i according to the equation formulated by Grynkiewicz et al. (22)
. [Ca2+]i determinations were done in parallel to the [Ca2+]m measurements on separate coverslips.
Confocal microscopy
Smooth muscle cells were transfected with a plasmid targeted to the same mitochondrial targeting sequence as for the mitochondrially targeted aequorin probe, but apoaequorin was exchanged for GFP in order to visualize the specific localization of the targeted probe (14)
. An Olympus BX50WI microscope using a 60x water dipping lens (numerical aperture of 0.90) and an Ultraview Nipkow confocal disk were used to generate the pictures shown in Fig. 3
. Data were acquired and analyzed with Ultraview 4.0 software.
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Electron microscopy
The primary fixative solution contained 1.5% glutaraldehyde, 1.5% paraformaldehyde, and 2% tannic acid in 0.1 M sodium cacodylate buffer, which was prewarmed to the same temperature as the experimental buffer solution (37°C). Cultured aortic smooth muscle cells were resuspended (with 0.25% trypsin and 1 mM EGTA for 1 min at 34°C) and transferred from the culture flask to a plastic centrifuge tube. After centrifugation at 1000 rpm for 5 min, the supernatant was quickly removed and replaced with the primary fixative solution. The fixed supernatant was cut into small blocks 1 x 0.5 x 0.2 mm in dimension and put in the same fixative for 2 h at 4°C on a shaker. The blocks were then washed three times in 0.1 M sodium cacodylate (30 min). In the process of secondary fixation, the blocks were put in 1% OsO4, 0.1 M sodium cacodylate buffer for 2 h, followed by three washes with distilled water (30 min). The blocks were further treated with 1% uranyl acetate for 1 h (en bloc staining), followed by three washes with distilled water. Increasing concentrations of ethanol (50%, 70%, 80%, 90%, and 95%) were used (10 min each) in the process of dehydration. 100% ethanol and propylene oxide were used (three 10 min washes each) for the final process of dehydration. The blocks were left overnight in the resin (TAAB 812 mix, medium hardness), then embedded in molds and placed in an oven at 60°C for 8 h. The embedded blocks were sectioned on a microtome using a diamond knife. The thickness of the sections was 80 nm. The sections were then placed on 400 mesh copper grids and stained with 1% uranyl acetate and Reynolds lead citrate for 4 and 3 min, respectively. Images of the cross sections of the muscle cells were obtained with a Phillips 300 electron microscope.
Statistical analysis
Where applicable, values are expressed as means ± SE; significance of difference was calculated by one-way analysis of variance and two-tailed Student’s t test for unpaired data in GraphPad Prism. Traces are representative of at least three experiments performed at least in duplicate. The off-line [Ca2+]i and [Ca2+]m data analysis was performed with the Matlab environment version 5.3 (MathWorks, Gümlingen, Switzerland) to determine the area under the curve (AUC). Microsoft Excel was used to generate figures.
Buffers
PSS contained in mM: HEPES, 5; KCl, 5; MgCl2, 1; NaCl 145; CaCl2, 1.2; glucose, 10. 0 Ca2+ solution was the same as PSS without CaCl2. 0 Na+ solution was the same as 0 Ca2+ solution except Na+ was replaced with an equimolar concentration of N-methyl-D-glucamine (NMDG). Osmolarity was measured for the latter solution with a vapor pressure osmometer (Wescor 5500, Logan, UT) and was 300 mOsm.
Chemicals
Coelenterazine and Fura-2 AM were from Molecular Probes (Eugene, OR). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was purchased from Fluka (Buchs, Switzerland). Thapsigargin (Tg), NMDG, adenosine triphosphate (ATP), [Arg (8)
vasopressin (AVP), nifedipine, 2-aminodiphenylborate (2-APB), cyclopiazonic acid (CPA), and U-73122 were from Sigma (St. Louis, MO). Digitonin was from Calbiochem (San Diego, CA). Effectene® was from Qiagen (Chatsworth, CA). Matrigel was from Fisher Scientific (Wohlen, Switzerland).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, C), although ATP stimulation produced a much larger response in these smooth muscle cells. 1 mM ATP was used to obtain highly reproducible results. Signals were obtained at lower concentrations but with less consistency. Thus, ATP potency seems to be diminished during culture. ATP increased [Ca2+]m to 4–6 µM whereas AVP produced increases of [Ca2+]m only up to 1–2 µM (see Fig. 5
for statistics). The [Ca2+]m responses were always transient. In contrast, ATP and AVP induced the ‘typical’ biphasic agonist induced increases in [Ca2+]i as measured by fura-2 fluorescence in parallel experiments in the same cell type (Fig. 1B, D
). However, cytoplasmic [Ca2+]i responses were similar for ATP and AVP.
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Role of VGCC and FCCP uncoupling
Nifedipine (1 µM) had no effect on the [Ca2+]m transient (Fig. 2
B). This suggests that the Ca2+ supplied to the MT comes from sources other than L-type VGCC. Preincubation with 2 µM FCCP in PSS 10 min before application of ATP (Fig. 2A
) or AVP (data not shown) completely inhibited the responses. This was not due to altered timing, as a time control revealed a robust ATP-induced [Ca2+]m transient after a 10 min preincubation in PSS (data not shown). It may therefore be concluded that the [Ca2+]m transient is mediated by the mitochondrial Ca2+ uniporter, which is dependent on an intact mitochondrial membrane potential. This result confirms specific localization of the mitochondrial aequorin plasmid shown by using a targeted MT-GFP. Confocal images in Fig. 3
clearly shows a vast mitochondrial network similar to data published in HeLa cells and convincingly demonstrates the correct localization of the mitochondrial-targeted probe.
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Effect of SERCA blockade on mitochondrial transients
The effects of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, CPA and Tg, were tested on agonist-induced [Ca2(+]m transients. Either 100 µM CPA or 1 µM Tg completely inhibited the ATP response (Figure 4
A, B). This implicates the SR as the source for the [Ca2+]m transient. Tg blockade without complete store depletion slightly enhanced the [Ca2+]m transient, possibly due to inhibition of Ca2+ reuptake by SERCA (Fig. 4C
). Tg or CPA alone caused a small and slow [Ca2+]m transient (Fig. 4B, C
). A summary of these results is shown in Fig. 5
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Role of IP3R
SR Ca2+ release by ATP is thought to be due to production of IP3 via activation of a G-protein and hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). Inhibition of phospholypase C (PLC), the enzyme that catalyzes the hydrolysis of PIP2 to IP3 and diacylglycerol, with 1 µM U-73122 partially inhibited the ATP-induced mitochondrial Ca2+ transient (Fig. 6
and Fig. 7
). This concentration is effective since the IC50 of U-73122 for inhibition of PLC has been reported to be 0.12 µM (23)
. The IP3 receptor (IP3R) inhibitor, 2-APB inhibited the [Ca2+]m response to the same extent as seen with maximal PLC inhibition with U-73122. Data are presented as AUC measurements analyzed as described in Materials and Methods. A summary graph for dose-dependent 2-APB and U-73122 inhibition of the [Ca2+]m transients induced by ATP is shown in Fig. 7
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Involvement of the Na+-Ca2+ exchanger
The above data show that mitochondrial Ca2+ uptake is initiated by Ca2+ release from the SR. When cells were exposed to Ca2+-free medium, the [Ca2+]m response to ATP decreased presumably because Ca2+ was lost from the SR (Fig. 8
A). However, this effect was not seen when, in addition to Ca2+, Na+ was removed from the bathing solution (Fig. 8B
). The results are summarized in Fig. 9
, which shows that the MT Ca2+ response declined by 50% in the absence of Ca2+. However, the response was not significantly attenuated when the NCX was blocked by Na+ removal.
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Electron microscopy
Electron micrograph images of fixed single smooth muscles cells from the same cell line show close appositions of the SR with the plasma membrane and MT (Fig. 10
B). The membranous structures in close proximity to MT shown in this electron micrograph are smooth, do not contain vesicles, and are not associated with stacks of rough endoplasmic reticulum. This indicates they are SR rather than part of the Golgi apparatus. Mitochondrial–SR junctional spaces are clearly visible (Fig. 10B, C
), supporting the notion of Ca2+ accumulation in this restricted space, which appears to be on the order of 20 nm in width. It is important to notice SR elements in contact with the plasma membrane (PM) and MT and separate regions of only MT–SR interactions in the deeper cytoplasm. Thus, two populations of MT can be distinguished: those associated with SR-PM elements and those located deeper in the cytoplasm interacting only with SR elements. The frequency of these MT–SR junctional spaces was calculated to be 73 ± 2% (standard error, SE) from 15 different cells, and the average distance between the MT and SR was 18.8 ± 0.8 nm (SE) calculated from an average of 20 cells.
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Based on the experimental evidence presented here, a model for the major Ca2+ signaling events in vascular smooth muscle is depicted in Fig. 11
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 nm wide gap between the SR and the MT, where the local Ca2+ concentration in this cytoplasmic domain ([Ca2+]MT-SR) would increase by 10 µM during opening of SR release channels.
A role for MT in Ca2+ regulation in smooth muscle is still controversial. However, there are many studies using a variety of techniques that, for the most part, show convincing evidence for an important role of MT in Ca2+ homeostasis. For example, in rabbit aortic smooth muscle, 45Ca2+ fluxes have shown that the MT take up Ca2+ when the extracellular K+ is increased (24)
. The fluorescent indicator rhod-2 (a lipophilic cationic dye) has also been used to measure mitochondrial [Ca2+] in pulmonary artery myocytes simultaneously with fura 2 measurements of [Ca2+]i. Results from this study show that Ca2+ release from the SR increases [Ca2+]m via the ryanodine receptors (RyR) and IP3R (25)
. Another low-affinity Ca2+-sensitive fluorescent indicator, mag-fura, which was thought to mainly localize in the SR, has been used in rabbit aortic myocytes and revealed compartmentalization of the dye in the MT in addition to SR elements. The authors report that mitochondrial inhibitors profoundly affected Ca2+ release from the SR and thereby suggest a functional integration between SR and MT in aortic smooth muscle (26)
. They show that mitochondrial Ca2+ remained elevated for several minutes after stimulation as opposed to [Ca2+]i, which showed a rapid decline on removal of the agonist. All of our agonist-mediated mitochondrial responses were transient. However, this discrepancy may be due to the different modes of [Ca2+]m measurement, rhod-2 vs. targeted aequorin, although experiments done with rhod-2 by Hajnoczky et al. agree with our targeted aequorin measurements (11)
. Another explanation for these varied results and maintenance of the MT response may reflect the lack of a Na+-dependent Ca2+ efflux pathway in the MT of some smooth muscles (27)
. Indirect evidence using classical pharmacological approaches suggests a role for MT in regulating Ca2+ in smooth muscle cells (28
, 29)
and guinea pig colonic smooth muscle (30)
. Finally, studies in A10 smooth muscle cells using the same targeted mitochondrial aequorin, but permeabilized with digitonin before experimentation, find that Ca2+-induced Ca2+ release (CICR) at the RyR generates microdomains of elevated [Ca2+] that are sensed by adjacent MT (31)
. These authors suggest that CICR is required for generating sufficient elevation of mitochondrial Ca2+. However, this study confirms data from many other cell types supporting the notion that MT can sense Ca2+ released directly from the ER or SR via IP3 channels. Therefore, release from IP3R and RyR may be important in producing the large, local increases in [Ca2+]MT-SR.
We present evidence here for a role of IP3R in directly supplying Ca2+ sensed by the MT uniporter. The PLC inhibitor U-73122 and the IP3R blocker 2-APB both caused about a 50% decrease in the ATP-induced [Ca2+]m transient (Fig. 6)
. Thus, it appears that half of the Ca2+ supplied to the MT is released from the SR through IP3R. The remaining Ca2+ is released from the SR, since SERCA blockade abolishes the entire [Ca2+]m transient. Presumably the RyR are involved in this process, although the mechanism is not clear at this time. Coupling between [Ca2+]m transients to RyR-mediated [Ca2+]i signals in smooth muscle cells has been reported by Drummond and co-workers (25)
. A study in which caged Ca2+ was released in smooth muscle cells from portal vein supports the concept of cooperativity between IP3R and RyR (32)
. Recent reports have shown that the second messengers cyclic-ADP-ribose and nicotinic acid adenine dinucleotide phosphate activate RyR (33
, 34)
. It is therefore possible that the MT may receive signals from release through RyR and IP3R, both of which may be in close spatial apposition to the MT uniporter.
This study shows that MT can sense activity of NCX by virtue of close apposition to the SR. Our results reveal that at 0 Ca2+ and low EGTA conditions, stimulation with 1 mM ATP generates a smaller transient response compared with control ATP applications. These responses do not diminish completely even after 1000 s in a 0 Ca2+ environment, but decline only to approximately half of a control ATP transient. On the contrary, substitution of Na+ by NMDG prevented any short-term decline in the ATP-induced [Ca2+]m transient (Fig. 8)
. We have previously shown that in vascular smooth muscle the NCX located at PM-SR junctions mediates transfer of Ca2+ between the SR lumen and extracellular space (35)
. If MT are located close to that portion of the SR that is depleted via the NCX, their responses would show a parallel decline. Thus, our results could be explained by assuming the existence of two distinct populations of MT: those associated with peripheral SR elements that form junctional complexes with the PM and other MT situated deeper in the cytoplasm that are associated with SR elements that do not form junctions with the PM. This hypothesis seems all the more plausible by examination of electron micrographs of our smooth muscle cells. In a single cell, SR–MT junctions are obvious, and the organellar membranes are separated by only 20 nm in many instances. Moreover, peripheral SR elements are observed that are in close contact with MT. Deeper in the cell, SR–MT junctions exist without any close association with the PM. Thus, the two populations of MT visible with the EM could possibly explain our functional findings.
After a prolonged period of exposure to Ca2+-free conditions, replenishment of Ca2+ was always accompanied by a smaller [Ca2+]m transient. This may be related to enhanced Ca2+ permeability of the PM due to activation of store-operated cation channels (SOCC) and nonspecific membrane destabilization (36)
. It is not likely that this [Ca2+]m transient was due exclusively to SOCC supplying MT, because SERCA blockade only transiently increased [Ca2+]m while the SOCC would remain activated. Whether the mitochondrial Ca2+ signal after Ca2+ replenishment is mediated by SR Ca2+ release remains to be investigated.
Finally, digitonin permeabilization at the end of all experiments often exhibited a biphasic response. This lends further support to the idea of two different populations of MT.
Although ATP and AVP produce almost equal increases in [Ca2+]i as measured by Fura-2, they show marked differences in Ca2+ accumulation by the MT. ATP shows a > 10-fold increase in [Ca2+]m compared with [Ca2+]i. On the other hand, AVP shows only a doubling of [Ca2+]m compared with [Ca2+]i. This apparent paradox could be explained by the fact that different agonists rely to different extents on Ca2+ influx and release in order to elevate [Ca2+]i. In this study, we have shown that [Ca2+]m is much more sensitive to release than influx. Thus, if the influx/release ratio were higher for AVP than for ATP, the mitochondrial Ca2+ signal would be proportionally smaller.
Although it is clear that MT accumulate Ca2+ on agonist stimulation, the functional significance of the mitochondrial Ca2+ signal remains to be established. MT contain many Ca2+-dependent enzymes (37
, 38)
; therefore, much of this Ca2+ may be used to signal production of ATP via oxidative phosphorylation. SR-mediated Ca2+ release can raise the mitochondrial [Ca2+], which in turn increases ATP generation to match the increased energy demands required for smooth muscle cell contraction. The MT may also act as a secondary buffer on receiving a signal from the SR. This additional buffering capacity of the cell may act to delay or spread the Ca2+ signal to the rest of the cell, as has been shown in pancreatic acinar cells (39)
. Furthermore, the close apposition between SR and MT membranes suggests SR–MT cross-talk in this restricted space. For example, in hepatocytes, mitochondrial Ca2+ uptake suppressed the positive effects of Ca2+ on the IP3R, reducing Ca2+ release at submaximal doses of agonist (40)
. The Ca2+ sensitivity of the IP3R is complicated, as a small increase in [Ca2+]i opens type I receptors, but micromolar increases in [Ca2+]i close type I IP3R (41)
and open type II and III receptors (42)
. In this way, buffering of the Ca2+ signal by mitochondrial uptake may act to enhance or restrict the evolution of [Ca2+]i signals (43)
. Mitochondria contain extrusion mechanisms, namely, a mitochondrial NCX (mNCX) and a Na+-independent pathway via H+/Ca2+ exchange that release the Ca2+ (albeit more slowly than the uptake of Ca2+ via the uniporter) back into the cytosol (44)
. However, emerging evidence using CPG-37157, a specific blocker of mNCX, in primary cultures of rat brain capillary endothelial cells suggests that the mNCX is the dominant extrusion mechanism. Blockade of the mNCX greatly enhanced the ATP-induced transient; the authors conclude that accumulation of Ca2+ by the MT is limited by the NCX, thereby allowing Ca2+ cycling to occur during [Ca2+]m transients (45)
. Once the MT have removed Ca2+ from the cytoplasm via the uniporter, they can return it more slowly to the cytoplasm to prolong the activity of high-affinity Ca2+-dependent processes and/or refill the SR by a secondary active transport (46)
. The mitochondrial permeability transition pore (PTP) may be critical in maintaining Ca2+ in the MT at reasonable levels. The PTP opens on high [Ca2+]m, which triggers a fast release of Ca2+ and has been implicated in the release of apoptotic factors. The PTP has been suggested to behave as a [Ca2+]i-activated Ca2+ release channel under certain conditions (44
, 47)
, which requires further investigation. Therefore, multiple mechanisms exist in which the MT play a role in Ca2+ homeostasis both within the MT itself and in shaping the spatio-temporal global Ca2+ signal via rapid uptake and slow release of Ca2+.
In conclusion, the existence of an MT–SR-restricted cytoplasmic domain in vascular smooth muscle cells allows accumulation of µM Ca2+ that is sensed by the mitochondrial Ca2+ uniporter (Fig. 11)
. Only release of Ca2+ from SR channels and not influx contributes to this large, local increase in Ca2+. Half of the MT are associated through the superficial SR with the PM and can be indirectly affected by the activity of the NCX. Therefore, mitochondrial Ca2+ regulation is dependent on the filling state of the SR and, as a result, functions of MT in smooth muscle are 1) dampening and prolongation of the cytoplasmic Ca2+ response, 2) refilling of the SR, and 3) regulation of oxidative phosphorylation.
Future experiments with other specifically targeted aequorins will provide further insight into the complex Ca2+ signals regulating vasoconstriction and relaxation in vascular smooth muscle. Finally, studies in single cells and intact tissues using specifically targeted molecular probes may lead to a better understanding of the important role of MT in Ca2+ signaling, and the development of new drug targets in conditions where mitochondrial Ca2+ regulation is disrupted, such as Duchenne’s muscular dystrophy (48)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 30, 2002.
Revision received September 5, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P < 0.05 compared with AVP.
