Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575
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ABSTRACT |
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Experiments were performed to identify the biophysical
properties of store-operated Ca2+ channels (SOC) in
cultured human glomerular mesangial cells (MC). A fluorometric
technique (fura 2) was utilized to monitor the change in intracellular
calcium concentration
([Ca2+]i) evoked by
elevating external [Ca2+] from 10 nM to 1 mM
([Ca2+]). Under control conditions,
[Ca2+] averaged 6 nM and was unaffected by
elevating bath [K+]. After treatment with 1 µM thapsigargin to deplete the intracellular Ca2+ store,
the change in [Ca2+]i
(
[Ca2+]th) averaged 147 ± 16 nM. In thapsigargin-treated MC studied under depolarizing conditions
(75 mM bath K+),
[Ca2+]th was 45 ± 7 nM. The
[Ca2+]th response of
thapsigargin-treated cells was inhibited by La3+
(IC50 = 335 nM) but was unaffected by 5 µM
Cd2+. In patch clamp studies, inward currents were observed
in cell-attached patches with either 90 mM Ba2+ or
Ca2+ in the pipette and 140 mM KCl in the bathing solution.
The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were
Ca2+ > Ba2+ >> K+. These
channels were sensitive to 2 µM La3+, insensitive to 5 µM Cd2+, and voltage independent, with an average channel
activity (NPo) of 1.02 at command
potential (
Vp) ranging from 0 to
80 mV. In summary, MC exhibited an electrogenic
Ca2+ influx pathway that is suggestive of Ca2+
entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on
estimates of whole cell Ca2+ influx derived from our data,
we conclude that SOC with low single-channel conductance must be highly
abundant in MC to allow significant capacitative Ca2+ entry
in response to depletion of the intracellular store.
cadmium; lanthanum; fura 2 fluorescence; patch clamp; thapsigargin; angiotensin II
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INTRODUCTION |
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HUMAN GLOMERULAR MESANGIAL cells (MC) have contractile properties similar to smooth muscle cells of the vasculature and other organs. In MC, angiotensin II (ANG II) evokes an initial inositol trisphosphate (IP3)-mediated release of calcium from intracellular stores and a secondary sustained elevation in intracellular calcium concentration ([Ca2+]i) due to entry of calcium into the cell through ion-selective channels. These ion-selective channels are voltage-gated (VGCC) (9, 28, 38) and have properties consistent with the L-type, dihydropyridine-sensitive, Ca2+-selective channels described in cardiac cells (18) and vascular smooth muscle (37).
In addition to VGCC, recent studies have shown that some smooth muscle cells (7, 26, 27) and MC (23) also possess store-operated calcium channels (SOC). Initially characterized in a variety of nonexcitable cells (1, 15, 19), SOC are activated upon release of calcium from intracellular stores. Elevated [Ca2+]i is rapidly reduced back toward baseline by the Na+/Ca2+ exchanger (21) and Ca2+-ATPase (plasma membrane calcium ATPase, PMCA) in the plasmalemmal membrane and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) (29). Efflux through the plasma membrane transporters yields a net loss of calcium from the cell. SOC activation provides a Ca2+ influx mechanism (capacitative Ca2+ entry) that, in concert with the action of SERCA, replenishes the intracellular Ca2+ store.
Although the store-operated macroscopic current has been studied extensively, the single-channel properties have varied depending on the cells of study. These findings may be the result of differential expression of splice variants or related to the multiple technical difficulties that arise when studying the single-channel properties of SOC. The single-channel currents are very small, and rundown occurs rapidly in excised patches. Moreover, it may be difficult to activate SOC in cell-attached patches given that physical proximity to the endoplasmic reticulum appears critical. Although blockers of SOC have been reported previously (5), a detailed pharmacological profile has not yet been established.
One method to distinguish types of calcium channels in cell membranes is to determine the differential blocking effects of other multivalent cations. Different types of calcium-selective channels are blocked by multivalent cations with varying potencies. For example, VGCC are more sensitive than receptor-operated channels to blockade by Cd2+ (32), whereas receptor-operated Ca2+ channels are more sensitive than VGCC to blockade by La3+. Although high concentrations of these multivalent cations will equally inhibit all Ca2+-selective channels, low concentrations can distinguish the activities of VGCC and SOC in MC.
Previous studies have described with fluorescence microscopy the presence of SOC in MC (23, 24); however, the biophysical properties have not been defined. The present study was performed to describe some of the properties of SOC in cultured MC and determine whether multivalent cations can be used to differentiate SOC from VGCC. Results of experiments using fura 2 to measure [Ca2+]i indicate that thapsigargin depletes intracellular stores of Ca2+ in MC and evokes a Ca2+ cell entry pathway that does not involve VGCC. Single-channel patch clamp experiments provide a description of a Ca2+-selective channel with biophysical properties consistent with SOC. Information regarding the blocking effects of these multivalent cations can be useful for distinguishing the functional roles of these channels during the response to a contractile agonist.
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METHODS |
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Mesangial Cell Cultures
Human MC were subcultured to no more than 10 generations by standard methods previously described (13). Cells were cultured in DMEM supplemented with 10 mM HEPES (pH 7.4), 2.0 mM glutamine, 0.66 U/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20% fetal bovine serum. Upon reaching confluence, cells were studied within 56 h of passage onto 22 × 22-mm cover glasses (Fisher, Pittsburgh, PA).Fura 2 Measurements of [Ca2+]i
Measurements of [Ca2+]i in MC using fura 2 were performed using dual excitation wavelength fluorescence microscopy as previously described (3, 8). In brief, [Ca2+]i was monitored in individual MC placed in a perfusion chamber (Warner model RC-2OH) mounted on the stage of a Nikon Diaphot model 300 inverted microscope. The cells were illuminated alternately at 340- and 380-nm wavelengths (3-nm bandwidths) with light provided by a Deltascan dual monochromator system (Photon Technology International, Monmouth Junction, NJ). To monitor fluorescence emission (510 nm, 20-nm bandpass) from a single cell, an adjustable optical sampling window was positioned within the light path prior to detection by a photon-counting photomultiplier. Background-corrected data were collected (5 points/s), stored, and processed using the FeliX software package (Photon Technologies). Calibration of the fura 2 signal was performed according to established methods previously described (3, 8). Cells were loaded with fura 2 by incubation for 70-90 min (20°C) in PSS (135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2 and 10 mM HEPES) containing 7 µM of the acetoxymethyl ester of fura 2 (fura 2-AM), 0.09 g/dl DMSO, and 0.018 g/dl Pluronic F-127 (Molecular Probes, Eugene, OR). In all experiments, the initial bathing solution contained (in mM) 135 NaCl, 5 KCl, 10 HEPES, and 1 CaCl2. In some experiments, the free Ca2+ concentration of the bath was adjusted to 1.0 µM by buffering with 1.08 mM EGTA, according to the calcium concentration program by MTK Software. Bath [Ca2+] was reduced to less than 10 nM by addition of EGTA.Patch Clamp Procedure
Patch clamp experiments were performed with the pipette attached to the membrane (cell attached). The bath solution contained (in mM) 140 KCl, 10 HEPES, and 1 CaCl2. The pipette solution contained either 90 mM CaCl2 or 90 mM BaCl2 plus 10 mM HEPES, pH 7.4.Single-channel analysis was made at 23°C using standard patch clamp
techniques (10, 34). The patch pipette, partially filled with solution,
was in contact with a Ag-AgCl wire on a polycarbonate holder connected
to the head stage of a patch clamp (model 501A; Warner Instrument,
Hamden, CT). The pipettes were lowered onto the cell membrane, and
suction was applied to obtain a high-resistance (>5 G) seal. The
unitary current (i), defined as zero for the closed state (C),
was determined as the mean of the best-fit Gaussian distribution of the
amplitude histograms. Channels were considered to be in an open state
(S) when the total current (I) was >(n
1/2)I and <(n + 1/2)I;
n is the maximum number of current levels observed. The open
probability (Po) was defined as the time spent in
an open state divided by the total time of the analyzed record. When
multiple channels occupied a patch, the channel activity was calculated
as NPo =
nPn,
where Pn is the probability of finding
n channels open. The Axoscope acquisition program and pClamp
program set 6.02 (Axon Instruments, Foster City, CA) were used to
record and analyze currents.
Thapsigargin was obtained from Calbiochem. Differences among groups of data were determined using the one-way ANOVA plus Student-Newman-Keuls test. P < 0.05 was considered statistically significant. Data are reported as means ± SE (n = number of cells).
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RESULTS |
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Fura 2 Experiments
Voltage dependence of thapsigargin-induced
Ca2+ entry pathway.
Thapsigargin, a potent and selective SERCA inhibitor (35), was utilized
in experiments designed to unveil and characterize capacitative
Ca2+ influx in MC. The effect of 1 µM thapsigargin on
[Ca2+]i is illustrated by the
tracing shown in Fig. 1A.
Thapsigargin typically evoked a transient increase in
[Ca2+]i to a peak value of 250 nM,
which subsided to achieve a sustained plateau at 100 nM. Subsequent
imposition of a decrease in bath [Ca2+] from 1 mM to <10 nM caused a decline in
[Ca2+]i to 35 nM, a situation that
promotes depletion of the intracellular store during SERCA inhibition.
When bath [Ca2+] was then restored to 1 mM,
[Ca2+]i rapidly increased to 110 nM. In contrast, MC not subjected to thapsigargin treatment displayed
very small responses to raising bath [Ca2+]. In
the typical tracing shown in Fig. 1B,
[Ca2+]i only rose from 29 to 33 nM
when bath [Ca2+] was increased from <10 nM to
1 mM in the absence of SERCA inhibition. Accordingly, the change in
[Ca2+]i evoked by raising bath
[Ca2+] from <10 nM to 1 mM in
thapsigargin-treated cells was viewed as an indication of
Ca2+ influx resulting from depletion of the intracellular
store (capacitative Ca2+ entry). In high-K+ (75 mM) solution, thapsigargin evoked an elevation in
[Ca2+]i from 50 to 250 nM, a value
similar to the response observed in PSS containing 5 mM K+.
However, the change in [Ca2+]i
caused by thapsigargin
([Ca2+]th)
increased by only 40 nM under depolarizing conditions (Fig. 1C). As shown in the summary bar graph of Fig. 1D,
[Ca2+]th was significantly
greater in both PSS (5 mM K+) and high KCl after
intracellular stores were depleted by the application of
thapsigargin. Furthermore, after thapsigargin-evoked store
depletion,
[Ca2+]th was greater
in the presence of 5 mM K+ than that observed under
depolarizing conditions (75 mM K+). In the absence of
thapsigargin,
[Ca2+]i was very
small and was unaffected by bath [K+]. These
data indicate that the thapsigargin-induced Ca2+ entry
pathway is affected by cell potential. This pathway is marked by an
inward flux of (positive) calcium ions evoked by the thapsigargin
depletion of intracellular stores. The Ca2+ entry pathway
is not likely VGCC, because the inward flux of Ca2+ was
decreased rather than exaggerated by membrane depolarization. Moreover,
the major component of this pathway is not
Na+/Ca2+ exchange, because in this study
Ca2+ entry is elevated with high external Na+.
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Use of multivalent cations to distinguish
Ca2+ influx through VGCC and
SOC.
Additional experiments probed the ability of multivalent cations to
block capacitative Ca2+ entry through SOC. Typical tracings
of [Ca2+]i and a dose-dependent
curve for the blocking effects of La3+ on
[Ca2+]th in thapsigargin-treated
MC are presented in Fig. 2. As shown in the
[Ca2+]i tracings of Fig.
2A, La3+ dose-dependently reduced
[Ca2+]th in thapsigargin-treated
MC. The dose-response curve (Fig. 2B) reveals a maximal
blocking concentration of La3+ between 2 and 20 µM and an
IC50 of 335 nM. Figure 3 shows
the effects of 5 µM Cd2+ on capacitative Ca2+
entry. As shown in the tracing of
[Ca2+]i (Fig. 3A) and the
summary bar graph (Fig. 3B), compared with La3+,
Cd2+ was a relatively ineffective blocker of
[Ca2+]th in thapsigargin-treated
MC.
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Patch Clamp Experiments
Patch clamp experiments were performed to determine whether MC contained Ca2+ channels with properties consistent with the capacitative Ca2+ entry pathway evident in the fura 2 experiments. Figure 5A shows single-channel (inward) current tracings of a cell-attached patch at various holding potentials. These channels were in groups of at least three per patch and were spontaneously active in cell-attached patches with either Ca2+ or Ba2+ as the charge carrier.
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Only inward currents were observed at command potentials
(Vp) ranging from 0 to
80 mV. The current-voltage relations (Fig. 5B) revealed
a 0.7-pS channel with calcium as the charge carrier and a 2.1-pS
calcium channel with barium as the charge carrier. The selectivity
values of 87 and 8.2 for Ca2+/K+ and
Ba2+/K+ were estimated by the
Goldman-Hodgkin-Katz equation, using reversal potentials of 123 mV
(Ca2+) and 63 mV (Ba2+), and assuming
[K+]i = 120 mM (14). The membrane
potential was near zero with 140 mM KCl in the bathing solution. As
shown in Fig. 5C, the NPo of these channels
did not change significantly when varying the holding potential.
The differential blocking effects of La3+ and
Cd2+ on the calcium channel are presented in Fig.
6. As shown in the tracings of Fig.
6A, addition of 5 µM Cd2+ to the pipette solution
containing 90 mM BaCl2 (Vp =
60 mV) did not affect NPo of
the calcium channels. However, when La3+ at concentrations
of 200 nM and 2 µM was added to the pipette solution, channel
activity diminished to 0.40 and 0.16, respectively. As shown in the
summary bar graph (Fig. 6B), there was no significant effect of
Cd2+ on NPo [5 µM
Cd2+, 0.94 ± 0.16 (n = 7) vs. control, 1.01 ± 0.06 (n = 13)]. However, 200 nM La3+
decreased NPo to a value (0.4 ± 0.06, n = 5) significantly lower than control, and 2 µM La3+
decreased NPo to a value (0.16 ± 0.06, n = 5) significantly lower than control and 200 nM La3+. The
degree of inhibition of the putative SOC in the patch clamp experiments
was 42% with 200 nM La3+ and 82% with 2 µM
La3+. These values were close to the percentage inhibition
of SOC by these concentrations of La3+ as determined by the
fura 2 experiments (38 ± 14% with 200 nM La3+ and 67 ± 10% with 2 µM La3+, see Fig. 2B).
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Thus the voltage independence and the sensitivity to block by La3+ > Cd2+ was distinct from VGCC and was consistent with the properties described for capacitative Ca2+ entry through SOC in the fura 2 experiments. Moreover, the similarities in the sensitivities to block by La3+ strongly suggest that the calcium channel in the patch clamp experiments is the same SOC described for capacitative Ca2+ entry in the fura 2 experiments.
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DISCUSSION |
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The results of this study revealed that MC in culture possess a Ca2+ cell entry pathway activated upon release of intracellular stores and reduced by depolarizing potentials. The properties of this pathway are consistent with the capacitative Ca2+ entry pathway through SOC previously described in several types of nonexcitable cells (30). The relative La3+ > Cd2+ sensitivity, low conductance, absence of voltage gating, and relative selectivity of Ca2+ > Ba2+ observed in MC using patch clamp methods is consistent with previously described properties of SOC in a variety of nonexcitable cells (2, 11) and distinct from the VGCC recently described in these cells (9).
Differentiating Ca2+ Influx Pathways with La3+ and Cd2+
The differential sensitivities of Ca2+ channels to La3+ and Cd2+ can be used to determine the roles of Ca2+ channels in cells. For example, although 5 µM Cd2+ did not inhibit SOC in the present study, it was previously shown that <10 µM Cd2+ is an effective inhibitor of L-type VGCC (25). We therefore determined whether 5 µM Cd2+ could inhibit VGCC of MC. Electrophysiological and fura 2 evidence has shown that MC contain L-type (dihydropyridine-sensitive) VGCC that are activated by depolarizing potentials and ANG II (9, 28, 38). As described by results from patch clamp studies, these channels are 11 pS with barium as the charge carrier and are steeply voltage dependent between potentials of 0 andThe failure of 5 µM Cd2+ to block the thapsigargin-evoked Ca2+ influx pathway in MC is consistent with the notion that VGCC does not have a role in replenishing calcium stores. Moreover, the lack of effect of 5 µM Cd2+ is consistent with previous findings by Hoth and Penner (12) that the IC50 for block of SOC by Cd2+ is 240 µM. Lanthanum, on the other hand, is a more powerful blocker of SOC than VGCC (12). The differential sensitivities of VGCC and SOC to these multivalent cations allows pharmacological assessment of capacitative Ca2+ entry through SOC.
Single-Channel Properties of SOC
Although several studies using fura 2 fluorescence have indicated the presence of SOC in a variety of cells (2), patch clamp studies have yielded disparate descriptions of the single-channel properties. There are several reasons for the difficulties in determining the properties of SOC. First, the single-channel conductance of SOC is relatively small and difficult to resolve with patch clamp analysis. Second, in excised patches, channel activity runs down within ~5 s (20). Third, the coupling mechanism for sensing store depletion by SOC is likely spatially restricted (2, 30) and disrupted upon obtaining a cell-attached patch in which the cell membrane is pulled into the patch pipette (33). Therefore, regulatory properties of SOC determined with whole-cell methods of analysis may not coincide with results from single-channel analysis.Earlier experiments utilized fluctuation analysis to estimate that the single-channel conductance of SOC is only 24 fS in 100 mM extracellular Ca2+ (39). However, the unitary conductance of SOC in A431 cells was measured at 2 pS with 200 mM CaCl2 and 10 pS with 80 mM BaCl2 (20). A more recent study determined that the conductance of SOC in Jurkat T lymphocytes was 36-40 pS when all Ca2+ was removed and Na+ was used as a charge carrier (15). On the basis of macroscopic Ca2+ current and the ratio of Na+/Ca2+ current, the single-channel current has been estimated to be as high as 1.6 pS with 20 mM Ca2+ as the charge carrier. Therefore, the single-channel conductances evident in MC (0.7 pS with Ca2+ as the charge carrier and 2.1 pS with Ba2+ as the charge carrier) are within the range of measurements obtained for SOC in A431 cells and Jurkat T lymphocytes.
The relative selectivity of Ca2+/K+ for the putative SOC in MC was estimated at 87. This value is based on the assumption that the membrane potential was close to zero in cell-attached patches and that [K+]i was 120 mM, as previously measured in rat MC (14). A high selectivity for Ca2+/K+ is consistent with the view that the SOC is highly selective for divalent over monovalent cations (12). Although the relative Ca2+/Ba2+ selectivity was not determined directly, the relative Ca2+/K+ selectivity of 87 was much larger than Ba2+/K+ (estimated at 8.2). The higher selectivity for Ca2+ relative to Ba2+ is unique to SOC and was described earlier with whole cell current measurements for SOC of mast cells (11) and lymphocytes (39). Despite the finding that the single-channel conductance is larger when Ba2+ is the charge carrier, the putative SOC in MC is more selective for Ca2+. Similarly, SOC of lymphocytes are very conductive for Na+ ions but only when divalent cations are removed from the external solution (15). Thus divalent cations have more affinity for the SOC pore but permeate more slowly.
While several studies have provided estimates for the single-channel conductance and selectivity of SOC, there is less information regarding the multivalent blocking properties of SOC. In the present study, 2 µM La3+ blocked completely the activity of the barium-selective current. However, 5 µM Cd2+ was an ineffective blocker of the putative SOC. The relative blocking order of La3+ > Cd2+ in the patch and fura 2 experiments is consistent with other studies that have shown a greater La3+ compared with Cd2+ micromolar block of SOC (12, 32). Although a complete dose response for La3+ block of the putative SOC in the patch clamp experiments was not determined, the partial block by 200 nM and the complete block by 2 µM coincides well with the fura 2 experiments, which indicated an IC50 of ~335 nM for the block of SOC by La3+. This is strong evidence that the putative SOC is the same pathway for the capacitative entry of Ca2+ detected in the fura 2 experiments.
Previous observations have demonstrated that the SOC is not voltage gated (11). However, when membrane potential was depolarized with high K+ in the present study, thapsigargin-evoked Ca2+ entry was significantly decreased. Considering that membrane voltage (Vm) could be depolarized by as much as 70 mV when changing from 5 mM to 75 mM K+, the decreased capacitative Ca2+ entry could be the result of decreasing the driving force for Ca2+ entry through SOC. Interestingly, a previous study found that membrane depolarization by high K+ or blockers of K+ channels inhibit the rise in [Ca2+]i through SOC evoked by T cell mitogens (17). Moreover, membrane hyperpolarization by the potassium ionophore valinomycin reverses the effects of K+ channel blockers (31). These results show that membrane potential plays a role in maintaining the driving force for sustained Ca2+ entry during store depletion. Maintenance of a hyperpolarizing membrane potential would not be critical for Ca2+ entry into the cell during store depletion, because the chemical gradient would still be very large.
The genetic equivalent of SOC, like the channels in native cells, has been difficult to assess in expression systems. Several studies have suggested that transient receptor protein (trp) of Drosophila photoreceptors is the SOC channel (2, 36). Expressed in Sf9 cells, trp was activated by thapsigargin (36). However, in HEK-293 cells, Htrp3 was expressed as 17-pS and 66-pS channels that were activated by depolarizing potentials and were nonselective to monovalent and divalent cations (16). Therefore, because many of the single-channel properties of expressed trp are different from those of SOC in native cells, the genetic equivalent of SOC remains uncertain(6).
Ca2+ Channels in MC
At least three other Ca2+ channels have been described in either rat or human MC in culture. The L-type VGCC (11 pS) was recently described in MC (9). Chen et al. (4) described a 21-pS Ca2+-permeable and mechanosensitive channel in rat MC, and Matsunaga et al. (22) found a 1-pS receptor-operated Ca2+ channel in rat MC. Although the single-channel conductance of 1 pS was close to that of the putative SOC, the divalent/monovalent selectivity for the receptor-operated channels was close to unity and not likely responsible for the highly Ca2+-selective currents evident in the present study. The putative SOC of the present study is therefore distinct from these previously described channels.On the basis of estimated surface area and an average of three channels
per patch, the total number of channels present in a single MC is
~86,000. This value can be compared with an estimate based on the
average rate of increase in [Ca2+]i
(2.4 nM/s) upon elevating [Ca2+]i
from <10 nM to 1 mM in the thapsigargin-treated cells. Using an
estimated cell volume, the equivalent Ca2+ current that
would account for such a rise in
[Ca2+]i would be ~205 pA. If the
channels were maximally activated to an average Po
of 0.15 and the channel amplitude were 0.3 pA (estimated for 1 mM
Ca2+ at a membrane potential of 40 mV), then the
number of channels per cell would be approximated at 47,000. This is a
minimum approximation because Ca2+ that enters the cell is
immediately buffered, thereby blunting the measured rise in
[Ca2+]i. These considerations
suggest that the putative low-conductance SOC must be highly abundant
in MC to provide the capacitative Ca2+ entry evident upon depletion.
In summary, we have described Ca2+ entry pathways using fura 2 and patch clamp methods of analysis. The fura 2 experiments revealed a capacitative Ca2+ entry pathway activated by thapsigargin-evoked depletion of intracellular stores and sensitive to blockade by La3+ over Cd2+. The patch clamp experiments disclosed a La3+-sensitive, Ca2+-selective current with voltage characteristics consistent with the SOC. Both measurements suggest that a typical mesangial cell contains at least 50,000 low-conductance SOC. Therefore, human glomerular MC, contractile cells previously shown to contain VGCC, contain an abundance of SOC, which likely function to replenish intracellular calcium after IP3-mediated release from the endoplasmic reticulum.
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ACKNOWLEDGEMENTS |
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We are grateful to Hanna Abboud of the University of Texas Health Science Center at San Antonio for providing us with cultures of human mesangial cells.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49561 (to S. C. Sansom). R. Ma was supported by a fellowship grant from the American Heart Association (Heartland Affiliate).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Physiology and Biophysics, Univ. of Nebraska Medical Center, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: ssansom{at}unmc.edu).
Received 23 September 1999; accepted in final form 17 January 2000.
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