February 2008
Volume 49, Issue 2
Free
Physiology and Pharmacology  |   February 2008
Activation of Store-Operated Ca2+ Channels in Trabecular Meshwork Cells
Author Affiliations
  • Elena Abad
    From the Laboratory of Neurophysiology, Faculty of Medicine, and the
  • Gisela Lorente
    From the Laboratory of Neurophysiology, Faculty of Medicine, and the
  • Núria Gavara
    Biophysics Unit, Department of Physiological Sciences I-Institute of Biomedical Investigations August Pi i Sunyer, School of Medicine, University of Barcelona, Spain.
  • Miguel Morales
    From the Laboratory of Neurophysiology, Faculty of Medicine, and the
  • Arcadi Gual
    From the Laboratory of Neurophysiology, Faculty of Medicine, and the
  • Xavier Gasull
    From the Laboratory of Neurophysiology, Faculty of Medicine, and the
Investigative Ophthalmology & Visual Science February 2008, Vol.49, 677-686. doi:10.1167/iovs.07-1080
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Elena Abad, Gisela Lorente, Núria Gavara, Miguel Morales, Arcadi Gual, Xavier Gasull; Activation of Store-Operated Ca2+ Channels in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(2):677-686. doi: 10.1167/iovs.07-1080.

      Download citation file:


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

      ×
  • Supplements
Abstract

purpose. In nonexcitable cells, Gq-coupled membrane receptor activation induces a biphasic increase in intracellular calcium ([Ca2+]i) expressed as an initial IP3-dependent release from intracellular stores followed by a sustained Ca2+ influx from the extracellular space that involves store-operated Ca2+ channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca2+ signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility.

methods. [Ca2+]i was measured in cultured bovine TM cells loaded with Fura-2. Ca2+ currents were recorded using the patch clamp technique. Cell contractility measurements were assessed by traction microscopy.

results. BK and ET-1 activate a store-operated Ca2+ entry that was greatly reduced in the absence of extracellular Ca2+ or by preincubation with SOC blocker 2-APB or SKF96365. Store-operated Ca2+ currents were also activated by intracellular dialysis with IP3 + EGTA or after stimulation with thapsigargin. Electrophysiological characterization supports the presence of Ca2+ release-activated Ca2+ channels (CRACs) and nonselective cation channels, of which TRPC1 and TRPC4 channels may be candidate TRPs detected in TM cells. Extracellular Ca2+ entry through SOCs is not required for TM cell contraction in response to BK or ET-1, but it modulates this process.

conclusions. Extracellular Ca2+ entry in TM cells in response to agonist stimulation and store-depletion is mediated by the activation of SOCs, which do not contribute to cell contraction but which may activate regulatory mechanisms to prevent excessive contraction. CRAC and TRPC channels involved represent interesting modulators of TM function to improve aqueous humor outflow.

The trabecular meshwork (TM) is a tissue located in the iridocorneal angle that actively regulates the flow of aqueous humor (AH) exiting the eye. The TM is a key determinant to maintain intraocular pressure, 1 and malfunction of this tissue often leads to ocular hypertension and glaucoma. 2 Among the different mechanisms that determine AH outflow pathway permeability, contraction and relaxation of TM cells appear to be among the most relevant. 3 In support of this, TM cells display contractile properties 4 and respond, through TM cell surface receptors, to substances that induce contractions, released by TM cells themselves or by surrounding tissues. In this sense, several substances known to contract the TM also reduce outflow facility, whereas cellular relaxation is commonly associated with the opposite effect. 3 5 6 7 8 9 In TM cells, tissue and cellular contraction have been reported after stimulation with carbachol, endothelin-1 (ET-1), or bradykinin (BK), among other substances. 6 10 11 Interestingly, in all cases, a biphasic increase in intracellular calcium concentration ([Ca2+]i) was observed when TM cells in culture were stimulated with the aforementioned substances. 6 12 13  
Free Ca2+ elevation is one of the early events occurring after the stimulation of many cell membrane receptors, and it is fundamental for cell survival and function. Commonly, this Ca2+ elevation is multiphasic and includes Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane. Extracellular Ca2+ entry is essential for many cellular processes such as cell growth and differentiation, cell motility, enzyme control, gene activation, exocytosis, nitric oxide (NO) production, and cell contraction. 14 15 The release of Ca2+ from intracellular stores by different mechanisms activates a store-operated Ca2+ entry (previously known as capacitative Ca2+ entry) that appears to be mediated by the coordinated interaction of STIM1 and Orai1 proteins; the former is the Ca2+ sensor and the latter is a plasma membrane calcium channel. 16 17 18 19 Agonists such as carbachol, signaling through Gq-coupled receptors, produce IP3-mediated depletion of Ca2+ stores that can activate store-operated Ca2+ channels (SOCs) but also, by means of STIM1, appear to activate TRPC1 channels in HEK293 cells. 20 TRPC1 is a member of the canonical transient receptor potential (TRPC) subfamily of channels that has been involved in receptor-operated Ca2+ entry after stimulation of the PLC pathway. 21 22 TRPC1, TRPC6, and other channels of their subfamily mediate the sustained Ca2+ increase that precedes Rho activation and endothelial cell contraction, 23 24 thus enhancing vascular permeability. 25  
It is possible to hypothesize that mechanisms similar to the ones described may be present in TM cells to regulate the permeability of the outflow pathway in response to different stimuli. Here we show that TM cells have two store-operated Ca2+ entry pathways, one involving Ca2+ release-activated Ca2+ channels (CRACs) and another likely involving TRPC channels. External factors activating Gq-coupled membrane receptors trigger a sustained Ca2+ increase that, though is not necessary for acute TM cell contraction, appears to modulate this process, probably in conjunction with other yet undetermined regulatory events. 
Materials and Methods
Culture of Trabecular Meshwork Cells
Bovine TM cell cultures were performed using eyes from 3- to 6-month-old cows obtained at the local abattoir 0.5 hour to 2 hours after death and kept in PBS at 4°C for not more than 1.5 hours. A slight modification of the technique described by Stamer et al. 26 was used. As described, 6 7 TM strips were digested with 2 mg/mL collagenase and 0.5 mg/mL bovine serum albumin (BSA) at 37°C for 2 hours. After trituration with fire-polished glass Pasteur pipettes, the supernatant was collected and centrifuged. The pellet was resuspended and seeded in culture flasks containing Dulbecco modified Eagle medium plus 10% fetal bovine serum, 100 mg/mL l-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B. Cells reached confluence 12 to 15 days later. Cell passages were performed using trypsin-EDTA. Cells from passages 1 to 3 were used. All products for cell culture were obtained from Sigma (Madrid, Spain). 
Cytosolic Free Ca2+ Measurement
Measurement of cytosolic free Ca2+([Ca2+]i) was performed as described in detail. 7 Briefly, bovine TM cells were plated on 25-mm diameter glass coverslips (VWR Scientific Inc., Philadelphia, PA) and then loaded with 5 μM fura-2/AM (Calbiochem, San Diego, CA) for 45 minutes at 37°C in incubation buffer (140 mM NaCl, 4.3 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, at pH 7.4 with NaOH). Ca2+ was omitted in the Ca2+-free solution, and 1 mM EGTA was added. Coverslips with fura-2–loaded cells were transferred to an open flow chamber (1 mL incubation buffer) mounted on the heated stage of an inverted microscope (IX70; Olympus, Tokyo, Japan) using a TILL monochromator as a source of illumination. Images were taken with an attached cooled charge-coupled device (CCD) camera (Orca II-ER; Hamamatsu Photonics, Hamamatsu, Japan) and were digitized, stored, and analyzed on a personal computer using image analysis software (Aquacosmos; Hamamatsu Photonics). After a stabilization period of 10 minutes, image pairs were obtained alternately every 4 seconds at excitation wavelengths of 340 (λ1) and 380 nm (λ2; 10-nm bandwidth filters) to excite the Ca2+-bound and Ca2+-free forms of this ratiometric dye, respectively. The emission wavelength was 510 nm (120-nm bandwidth filter). Typically, 10 to 20 cells were present in a field, and [Ca2+]i values were calculated and analyzed individually for each single cell from the 340- to 380-nm fluorescence ratios at each time point. 7 Drug responses in each field were homogeneous, and several experiments with cells from different primary cultures were used in all the groups assayed. 
Patch Clamp Procedures
TM cells were plated onto small glass coverslips and studied between 24 to 72 hours thereafter. Coverslips were transferred to a special chamber (0.2 mL) in the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) so the recordings could be performed. External solutions were superfused at a rate of 3 to 4 mL/min by gravity. Before the recording session was started, the culture medium was replaced with a bath recording solution. Recordings were performed at room temperature. Borosilicate glass patch pipettes were pulled in an electrode puller (P-97; Sutter Instruments, Novato, CA) with a filled-tip resistance between 7 MΩ and 9 MΩ. Pipette capacitance to ground was neutralized after the seal was formed. An Ag/AgCl ground electrode mounted in a 3 M KCl agar bridge was used. Positive pressure was applied before the pipette entered the bath and until cell contact. Whole-cell currents were recorded using a patch clamp amplifier (L/M-EPC7; Heka, Lambrecht/Pfalz, Germany), as described. 7 27 Data acquisition and command potentials were controlled with patch clamp software (pClamp 9.0; Axon Instruments, Sunnyvale, CA) using a digitizer (Digidata 1320A; Axon Instruments). After breaking into the whole-cell configuration, cells were allowed to stabilize and dialyze for 3 to 4 minutes before recording was begun. Whole-cell currents were recorded at 10 kHz. Cells were clamped at 0 mV, and 500-ms ramps from −100 to +100 mV were applied every 5 seconds. Data were corrected for the calculated theoretical junction potential values for each solution with the Junction Potential Calculator feature of the patch clamp software (pClamp 9.0; Axon Instruments), which uses the Henderson equation to calculate the liquid junction potential. Development of the current was assessed from the current amplitudes at potential of −80 mV recorded during voltage ramps. The first three ramps of the protocol or the three previous to the addition of the drug were used for leak subtraction in the subsequent current records. 
In the whole-cell perforated patch technique, pipettes were filled back with intracellular solution in which a polyene antifungal (Nystatin, 150 μg mL−1; Sigma) was dissolved. The solution was from a stock solution (3 mg/60 μL dimethyl sulfoxide) and sonicated to the final concentration. Slowly developing capacitative transients indicated establishment of the whole-cell perforated patch configuration. We continuously monitored series resistance to avoid sudden drops in this parameter, indicating the ruptured-patch configuration was achieved. Only experiments with constant series resistance during the whole recording were considered for analysis. 
The intracellular pipette solution contained 145 mM CsMethanesulfonate, 8 mM NaCl, 1 mM MgCl2, 6 mM MgATP, 0.2 mM EGTA, 10 mM Hepes (pH 7.2) adjusted with CsOH. In some experiments, 10 EGTA was used, as indicated. The standard isotonic bath solution contained 145 mM NaCl, 2.8 mM KCl, 10 mM CaCl2, 2 mM MgCl2, 10 mM CsCl, 10 mM glucose, 10 mM Hepes (pH 7.4) adjusted with NaOH. In some experiments, divalent-free solution (DVF) was used, as follows: 145 mM NaCl, 2.8 mM KCl, 10 mM CsCl, 2 mM EGTA, 10 mM glucose, 10 mM Hepes (pH 7.4) adjusted with NaOH. 
Western Blot Analysis
SDS-PAGE was performed using the Laemmli method. Samples were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) according to the standard procedure. Nonspecific protein-binding sites were blocked with a solution containing 2% BSA and 0.1% Tween-20 in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.4] and 137 mM NaCl; TBT-BSA) for 1 hour. Membranes were then incubated with rabbit anti-TRPC1, anti-TRPC3, anti-TRPC4, anti-TRPC5, anti-TRPC6 IgG (Alomone Laboratories Ltd., Jerusalem, Israel) at 1:200 in TBT-BSA for 1 hour. Membranes were washed three times with TBT-BSA and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibodies at 1:2500 (Jackson ImmunoResearch Laboratories, West Grove, PA) in TBS-T (0.1% Tween-20 in TBS) for 1 hour. Finally, the membranes were washed three times in TBS-T and twice in PBS. Detection was performed by a chemiluminescence method with substrate (Immuno-HRPTM Star Substrate; Bio-Rad). 
Measurement of Cell Contraction
Measurement of cell contraction was assessed by traction microscopy, as previously described in detail. 28 Briefly, cells were seeded in thin collagen-coated polyacrylamide gel disks, as described by Pelham and Wang. 29 Green fluorescent latex beads 0.2 μm in diameter were mixed with 2% acrylamide and 0.3% bis-acrylamide solution (1:125 vol/vol bead solution volume of acrylamide mixture). Gel disks approximately 70 μm thick and 8 mm in diameter attached to a glass coverslip were prepared with 10 μL of this solution and subsequently coated with 3 μg/cm2 collagen I. 
Coverslips containing cells cultured on polyacrylamide disks were mounted on the stage of an inverted fluorescence microscope (Eclipse TE2000; Nikon) placed on a vibration isolation table (Isostation; Newport, Irvine, CA). Bright-field and fluorescence images were acquired with a 12-bit resolution cooled CCD camera (Orca; Hamamatsu Photonics). The apparent pixel size after magnification (40×) was 0.16 μm, with a resultant field of view of 161 × 161 μm2. A bright-field image of an isolated cell was captured to determine its boundary. Subsequently, the apical surface of the gel was focused, and fluorescence images of the microbeads embedded near the surface of the gel were acquired at 1 image/min. After 5 minutes of baseline recording, BK or ET-1 was added, and fluorescent images were acquired for an additional 10 to 20 minutes. At the beginning of the recording, a bright-field image was captured to define the area of the cell. At the end of the experiment, the cells were removed from the gel by exposure to trypsin. Finally, an additional fluorescent image was recorded to determine the position of the beads in the unstrained gel (reference image). A small percentage of isolated cells showed partial detachment from the substrate after agonist addition. Detached cells were discarded for traction microscopy measurements. 
Cell boundary was determined using a Sobel edge detector algorithm implemented in a graphics program (LabView; National Instruments, Austin, TX). The projected area of the cell was computed as the area enclosed by the cell boundary. To compute traction forces (T) exerted by the cell on the substrate, 28 the displacement field of the gel substrate was first determined from the stored fluorescent bead images. The displacement field between each fluorescence image and the reference image was computed using the image correlation method. Spatial resolution of the displacement and traction maps was 1.3 μm. For each traction field, the total force magnitude (F) was computed by integrating the magnitude of T(x, y) over the projected area of the cell. Although the net vectorial force over the contact area was zero, the integral of the modulus provided a useful index of the cell contractile strength. The average traction of the cell was computed as F/cell area. 
Statistical Analysis
Data are presented as mean ± SEM and were analyzed using paired or unpaired t-tests and statistical analysis software (Prism 4.0; GraphPad, San Diego, CA). Two-tailed tests were used, with statistical significance set at P < 0.05. 
Results
Characterization of Extracellular Ca2+ Entry in TM Cells
Ca2+ signaling and homeostasis are important regulators of many processes, triggering proliferation, cell contraction, secretion, and information processing in many cell types. 15 Several substances in the aqueous humor can activate Gq protein-coupled receptors in the membrane of TM cells to elicit an increase in intracellular Ca2+. The pathways involved in this Ca2+ increase are still unknown in these cells. 6 7 30 31 32 33 34 To first investigate the ionic mechanisms allowing extracellular Ca2+ to enter TM cells, we compared BK and ET-1 responses in physiological medium (control) or Ca2+-free medium or the presence of 2-APB, an alleged blocker of store-operated Ca2+ entry. 35 Ca2+ increase induced by BK or ET-1 (Figs. 1A 1B)was attributed to an initial release of Ca2+ from intracellular stores followed by a sustained Ca2+ entry from the extracellular space that was impaired in Ca2+-free medium or in the presence of 50 μM 2-APB (preincubated for 2–3 minutes). These differences were quantified as a reduction in the time required for the Ca2+ transient to decrease by 70% from its maximal value (T70). Figure 1Cshows the difference in the recovery time among the different experimental groups. Extracellular Ca2+ entry also contributes to total peak amplitude that was smaller in Ca2+-free medium or with 2-APB (Figs. 1A 1B)
To clearly distinguish between the two phases that contribute to Ca2+ elevation, a second protocol was used, cell stimulation with 1 μM BK in Ca2+-free conditions, which caused a transient increase in intracellular Ca2+ representing Ca2+ release from intracellular stores. After the return to basal level, Ca2+ reintroduction elicited a second peak resulting from Ca2+ entry through open membrane channels (Fig. 1D) . This extracellular Ca2+ entry could be partially inhibited in the presence of 2-APB and SKF96365 (54% and 60% respectively; P < 0001), alleged blockers of several SOCs (Figs. 1D 1E) . 35 36 37 La3+ has also been described as a blocker of some of the channels that may contribute to Ca2+ entry. A slight decrease in the extracellular Ca2+ entry was observed when tested, but it was not significant compared with the control (27%; P > 0.05; Figure 1E ). 
We further investigated this extracellular Ca2+ entry pathway by studying Ca2+ currents across the cell membrane using the perforated-patch configuration with recording solutions for SOC detection (10 mM Ca2+ external solution). Whole-cell currents were recorded using voltage ramps from −100 to + 100 mV applied every 5 seconds. Stimulation with BK (1 μM) activated an inward current (Fig. 2A ; mean of 10 cells) that slowly developed over several minutes (−0.53 ± 0.18 pA/pF at −80 mV after 300 seconds). Current-to-voltage (I-V) relationships before (a) and after (b) BK stimulation are shown in Figure 2B . The current activated in the presence of BK increased at depolarized and hyperpolarized potentials compared with the baseline current, showed a slight inward rectification at hyperpolarized potentials, and presented a reversal potential near +6 mV. These experiments were performed in the presence of tetraethyl ammonium (TEA; 10 mM) to block outward potassium currents that could be activated by depolarization (Fig. 2B) . Similar I-V curves were obtained in the absence of TEA, but larger outward currents were present probably because of the outflow of K+ or Cs+ ions (not shown). It has been reported that Ca2+ release-activated Ca2+ currents (ICRAC) show a prominent inward rectification and a very positive reversal potential close to +60 mV. 14 A common observation in other cell types is the activation of ICRAC currents together with other nonselective cation currents. 38 In this scenario, I-V curves are almost linear with a slight inward rectification at hyperpolarized potentials, similar to the ones activated by BK in TM cells (Fig. 2B)
In addition to the electrophysiological recordings, the presence of putative nonselective cation currents was assessed by Western blot. Given that members of the canonical subfamily of TRP channels have been proposed to function as SOCs, 21 22 we tested for their presence in TM cells in culture. TRPC1 and TRPC4 proteins were detected, whereas TRPC6 levels were undetectable in bovine TM cells (Fig. 2C , upper panel). Protein bands were absent when antibodies were preincubated with their antigens. As a positive control, the same channel proteins were found in rat brain extracts. No clear bands were detected at the appropriate molecular weights for TRPC3 and TRPC5 with the antibodies used, though the presence of these channels in TM cells cannot be totally discarded. In addition to the studies in cultured cells, we also tested bovine TM tissue extracts for the presence of TRPC channels. In the native tissue, TRPC1 and TRPC4 were also found, but TRPC6 was not detected (Fig. 2C , lower panel). 
Store-Operated Calcium Entry in TM Cells
To study whether TM cells present a store-operated calcium entry activated by the depletion of intracellular stores without the participation of Gq-coupled receptors, we measured intracellular Ca2+ transients activated by thapsigargin (TG), a blocker of the endoplasmic reticulum Ca2+-ATPase pump that depletes intracellular Ca2+ stores. TG (1 μM) induced a Ca2+ increase in TM cells that lasted several minutes, with a T70 of 190.8 ± 21.2 seconds (Figs. 3A 3B) . Again, a TG-induced Ca2+ increase was greatly reduced in Ca2+-free solution (T70 = 125.1 ± 10 seconds; P < 0.01) or in the presence of 50 μM 2-APB (T70 = 112.04 ± 7.2 seconds; P < 0.05), thus showing that the release of intracellular Ca2+ in TM cells activated an extracellular Ca2+ entry without the involvement of membrane receptors. To corroborate these findings, a protocol similar to the one previously used with BK was used here. In all the cells tested (n = 40), after an initial Ca2+ transient in Ca2+-free medium, TG induced a second Ca2+ increase when extracellular Ca2+ was reintroduced (Fig. 3C) . SOC blocker 2-APB and trivalent cations such as La3+ and Gd 3 have been reported to inhibit TG-activated Ca2+ entry. 14 When we tested 2-APB at 50 μM by incubating it just before reintroducing extracellular Ca2+, the second Ca2+ peak was greatly blocked (62.1%; P < 0.001; n = 18; Figure 3D ). Similar inhibitions were found when 20 μM La3+ or Gd3+ were assayed (51.7% and 44.6%; P < 0.001; n = 60 and 17, respectively; Figs. 3E 3F ), which also indicated the presence of SOCs in the membranes of TM cells. 
SOCs can be activated by any procedure that empties the intracellular Ca2+ stores. 14 We used the whole-cell patch clamp technique to record the activated membrane currents using two different protocols to deplete intracellular stores. First, using the same protocol as experiments shown in Figure 2and after recording baseline currents, cells were challenged with 1 μM TG (+0.2 mM EGTA). When this drug was applied, a slowly developing inward current activated that was more prominent at hyperpolarized potentials (−1.11 ± 0.20 pA/pF at −80 mV after 460 seconds; Fig. 4A ; n = 6). This type of current is characteristic of CRAC channel activation, as described. 39 40 The I-V relationship showed a clear inward rectification, and the current asymptotically approached to zero at very positive voltages (> +60 mV) without a clear reversal potential, which indicates a high selectivity for Ca2+ (Fig. 4B) . In a series of parallel experiments, using a more physiological stimulus, Ca2+ store depletion was achieved by dialyzing the cell with an intracellular solution containing 30 μM IP3 (+10 mM EGTA). Under these conditions and at −80 mV, an inward current (−1.77 ± 0.53 pA/pF after 460 seconds; n = 6) activated in an exponential manner (τ = 194 seconds), lasting for several minutes until the end of the recording protocol (Fig. 4C) . Interestingly, the I-V relationship was different from the one obtained in TG experiments: inward rectification was not as prominent, the current reversed at +19 mV, and the outward current was observed at depolarized potentials, though it was smaller than the inward current (Fig. 4D) . This I-V relationship was similar to the one obtained when BK was used (Fig. 2) . It may be that more than one conductance—probably ICRAC and nonselective cation channels—was activated, as described in other cell types. 38 Control recordings were carried out by omitting IP3 from the pipette intracellular solution (+0.2 mM EGTA, as in TG experiments). Under these conditions, no current activation was observed (−0.16 ± 0.20 pA/pF; n = 5), further confirming that store depletion is required for activation of SOCs in TM cells (Fig. 4C , control). 
An important characteristic of SOCs is their Ca2+ selectivity, but in DVF, these channels become permeable to monovalent cations. We tested this feature by activating the current in the presence of IP3 + EGTA (as in previous experiments) and then switching to a DVF. As reported in other cell types, 41 a 2.2-fold increase in the current was found in DVF (P < 0.01; Figure 4Eleft; see example in Fig. 4F ) which was significantly blocked in the presence of 100 μM 2-APB (58%; P < 0.01 vs. current in DVF). We also tested the blocking effects of trivalent cations on the activated current by applying La3+ or Gd3+ (100 μM). A significant reduction in currents was observed for both cations (P < 0.05; Fig. 4E , right) with La3+ more effective (35%) than Gd3+ (19%). These results further confirm that TM cells present SOC channels with characteristics of ICRAC currents described in other cell types. 14  
Contribution of Extracellular Ca2+ Entry to TM Cellular Contraction
Several substances, such as BK, ET-1, or carbachol, induce cell or tissue contraction in the TM, which likely contributes to decreased outflow facility. 6 31 42 43 The contribution of extracellular Ca2+ entry after SOC activation was investigated by measuring single-cell contractions in culture using the traction microscopy technique that allows measurement of traction forces exerted by cells on a deformable substrate. 28 Therefore, we measured the force exerted by TM cells in culture before and after a BK or an ET-1 (1 and 0.1 μM, respectively) challenge. BK application induced a progressive increase in cell contraction that reached a maximum within 5 minutes (53% increase vs. baseline; P < 0.001; n = 7; Figs. 5A 5B ). A bigger contraction force was found when challenging cells with ET-1, which induced a maximal 202% increase over baseline contraction (P < 0.001; n = 7; Figs. 5C 5D ). Interestingly, when Ca2+ was omitted from the extracellular solution, the same drugs induced cell contractions that were even bigger and developed more quickly (compare contraction time-course in control and Ca2+-free medium in Figs. 5A and 5C ). No differences were found between the baseline force values in the presence or in the absence of Ca2+ (262.5 ± 48.2 vs. 235.4 ± 63; arbitrary units, n = 11 and n = 16 cells, respectively). These results show that extracellular Ca2+ entry may not be necessary to achieve full TM cell contraction after stimulation with drugs through the Gq-coupled receptors. In the same experiments performed in Ca2+-free medium, this ion was reintroduced at minute 12. In BK experiments and after Ca2+ reintroduction, contraction was still increased compared with baseline (P < 0.05) and did not show statistical differences compared with the period in Ca2+-free conditions (Fig. 5B) . Similar results were found in ET-1 experiments in which the reintroduction of Ca2+ did not further modify cell contractions (Fig. 5D) . Finally, we tested whether preincubation with 100 μM La3+ (a blocker of ICRAC currents that did not significantly block BK-stimulated extracellular Ca2+ entry [Figs. 1E 3E 3F 4E ]), was able to modify cell contraction in the presence of extracellular Ca2+. As shown in Figure 5B , La3+ did not reduce cell contraction, similar to what occurred in Ca2+-free medium. 
Discussion
Results of this study demonstrate that TM cells activate two different types of store-operated Ca2+ channels in response to agonist-mediated Gq-coupled receptor stimulation. The first SOC identified in TM cells shows electrophysiologic and pharmacologic characteristics of CRAC channels (Ca2+ release-activated Ca2+ channels) after TG-induced store depletion (Figs. 4A 4B) . 14 44 Similarly, evidence for CRAC channels has also been reported in monkey TM cells after store depletion with TG. 45 Recently, molecular counterparts of CRAC channels have been identified as composed of Orai1 (the pore-forming unit) and STIM1 (the endoplasmic reticulum Ca2+ sensor). 16 17 18 19 Heterologous expression of one of these proteins alone is insufficient to produce functionally active Ca2+ currents, but their coexpression reconstitutes SOC function, which demonstrates that these two proteins are essential and sufficient components of CRAC currents. 46 Loss of Ca2+ from intracellular stores promotes the mobilization of STIM1 close to the cell surface, where it interacts with Orai1 and permits extracellular Ca2+ entry. 18 47  
Interestingly, when store depletion in TM cells was stimulated by bradykinin or by cell dialysis with IP3 + EGTA, extracellular Ca2+ entry appeared to be mediated by more than one current. Although CRAC currents show a clear inward rectification and almost no reversal potential (Fig. 4) , activation of other nonselective currents with CRACs results in an almost linear I-V relationship, with the current reversing between +10 to +20 mV (Figs. 2B 4D) , as observed in other cell types. 38 La3+ results also indicate this because the blocking effect appears stronger on TG-mediated SOCs and weaker when SOC activation is mediated by agonist-receptor activation (Figs. 1 3) . In the latter situation, a more prominent activation of TRPs, which are less selective for Ca2+, is assumed. It is conceivable that the nonblockable parts of the Ca2+ responses arose from the participation of different channels and not from differential blocker sensitivity of the channels. Several studies have proposed that members of the TRPC channel family behave as SOCs because their activation after stimulation of G-coupled membrane receptors produces an extracellular Ca2+ entry. 48 49 50 51 TRPC1, TRPC4, and TRPC5 appear to be the most likely candidates to perform as SOCs, whereas TRPC3 and TRPC6 indirectly function as SOCs when they heteromultimerize with other TRPC channels. 52 In particular, TRPC1 and TRPC4 expression has been found in bovine TM cells (Fig. 2C) , and the mRNA for TRPC4 and TRPC6 has recently been reported in porcine cells (Li G, et al. IOVS 2007;48: ARVO E-Abstract 2066), which supports the hypothesis that members of this family of channels are activated on store depletion or by means of the Gq-PLC-PKC pathway, thus behaving as SOCs. In fact, two recent reports link the activation of CRAC channels and TRPC channels by means of STIM1, which is able to interact with Orai1 and with TRPC1, TRPC4, and TRPC5 to determine their function as SOCs. 20 52 Therefore, a common mechanism stimulates different membrane channels (CRAC and TRPCs) to act as SOCs and permits the entry of extracellular Ca2+. Our electrophysiological and Ca2+ imaging results after BK application or cell dialysis with IP3 are in agreement with such a scenario. In addition to BK and ET-1, compounds such as ATP, 7 histamine, 53 and carbachol 12 among others, induce similar Ca2+ mobilization involving SOCs. Unfortunately, channel characterization remains a challenge because specific blockers for each subtype of SOCs are lacking. The poor selectivity of the SOC blockers available did not allow us to reach strong conclusions about the channel subtypes present, but the differential effects of La3+, depending on the SOC activator, can reflect the different pharmacologic profiles of the channels involved, namely CRACs and TRPCs. Nevertheless, care should be taken when interpreting the results reported because, despite the proved effect in different SOC channels, 35 37 2-APB has inhibitory effects on currents through connexins 54 and IP3 receptors. In addition, SKF96365 is widely used to study SOC-mediated currents but is selectivity limited. 36 In the present study, though we used the minimum concentration necessary and short incubation times to minimize the poor selectivity of these compounds, undesired side effects could not be completely ruled out. 
Although the initial function proposed for store-operated Ca2+ entry was to replenish emptied stores, it was later involved in other cellular processes. 44 In addition, store-operated Ca2+ entry has been linked to agonist-induced cytoplasmic Ca2+ oscillations, which in turn encode information to evoke cellular responses, including exocytosis, mitochondrial ATP production, and gene transcription. 55 Interestingly, Ca2+ entry through SOCs, but not Ca2+ release from the endoplasmic reticulum, is an effective regulator of Ca2+-sensitive adenylate cyclase, suggesting that an intimate spatial relationship between SOCs and the these enzymes must exist. 56 Similar results have been found for endothelial nitric oxide synthase in COS-7 cells 57 and Ca2+-dependent phospholipase A2 in mast cells, where local Ca2+ entry through CRAC channels triggers arachidonic acid production and leukotriene C4 secretion. 58 Finally, Ca2+ entry in nonexcitable cells is an important signal to activate cell growth and proliferation, thus regulating gene transcription through a variety of transcription factors, including NFAT, NFκB, and AP-1 and early genes such as c-fos. 58 59  
Another important cellular event linked to Ca2+ is cell contraction. 15 This is especially important in the TM because contractile tone equilibrium is an important regulator of outflow facility. 3 To assess the role of receptor-operated Ca2+ entry on TM cells, we studied cellular contraction by using traction microscopy, a cellular technique that allows the measurement of contractile forces exerted by a cell attached to a deformable substrate. 28 Our results show that TM cells contract in response to BK or ET-1, as previously reported. 6 31 42 43 Interestingly, the release of intracellular Ca2+ is sufficient to induce agonist-mediated cell contraction because contraction can be elicited in the absence of extracellular Ca2+ (Fig. 5) , thus confirming previous results found in whole tissue. 31 60 Similar effects were reported with muscarinic agonists in TM strips, though in the total absence of Ca2+ (extra and intracellular), carbachol was unable to contract the tissue. 31 Nevertheless, even in the total absence of Ca2+, the phorbol ester phorbol 12-myristate 13-acetate (but not carbachol) is still able to slightly contract the cells, 60 an effect that can be blocked with a Y-27632, a Rho-kinase inhibitor. 
A growing body of evidence has shown that the Rho/Rho-kinase pathway is involved in TM cell contraction and outflow facility regulation. 9 43 60 61 62 Agonists such as ET-1, thromboxane A2 mimetics, and angiotensin II induce myosin light chain (MLC) phosphorylation in TM cells through the activation of Rho GTPase. 9 It appears clear that many external factors, after interacting with G-protein-coupled receptors, regulate the activity of Rho/Rho-kinase pathway and other intracellular mechanisms, such as Ca2+, to influence the phosphorylation status of MLC and, therefore, the contractile state of the outflow pathway, as has been demonstrated for ET-1. 9 By comparing the time-course of cell contraction in the presence or absence of Ca2+ (Figs. 5A 5C) , it appears that extracellular Ca2+ entry is not necessary for cell contraction but modulates this process probably by promoting the release of relaxing substances such as NO, as described in other cells. 63 Relaxing mechanisms would, therefore, counterbalance the contraction process triggered by the agonist. As previously mentioned, Ca2+ entry through SOCs has been shown to activate adenylate cyclases, NO synthase, or PLA2, which contributes to the relaxing effects. 56 Among the possible mechanisms possibly activated, cAMP or cGMP production has been shown to relax TM cells and to increase outflow facility. 64 65 66 67 In fact, in addition to increasing TM cell intracellular Ca2+, BK induces the production of PGE2 68 69 and enhances the effect of PGE2 on cAMP production. 70 It seems plausible that cAMP production is enhanced by extracellular Ca2+ entry stimulation of adenylate cyclase, as reported in other cell types. 56 Interestingly, BK effects on outflow facility appear to be biphasic, with an initial acute decrease 6 followed by a slowly developing and prolonged increase of outflow facility. 71 Again, extracellular Ca2+ entry induced by BK may be involved in PGE2 production, which mediates the increase in outflow facility. Other relaxing effects may be also activated by extracellular Ca2+ entry through SOCs. In particular, Ca2+-activated potassium channels are activated by several second messengers in TM cells as a mechanism to prevent excessive cell contraction. 7 27 65 72 Therefore, sustained Ca2+ entry activates these channels together with other mechanisms to reduce tissue contractility, as shown in human vascular endothelial cells in which Ca2+ store depletion and extracellular Ca2+ entry are required to trigger NO production by histamine or ATP. 73 This process is tightly regulated by Ca2+-activated potassium channels, which are essential for NO-mediated vasorelaxation. 73 Therefore, receptor- and store-operated Ca2+ entry may not be necessary for cell contraction in TM cells but may contribute to modulate cell contractility or to release different substances that regulate outflow facility as a local homeostatic mechanism. Future experiments will test this hypothesis and whether these mechanisms may be pathologically altered by chronic exposure to ET-1, TGF-β, or substances elevated in glaucoma. 
 
Figure 1.
 
Agonists linked to Gq-coupled receptors activate a store-operated Ca2+ entry. (A) Representative Ca2+ increase after BK (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Experiments similar to these are shown in (A) with endothelin-1 (ET-1, 0.1 μM). (C) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated by each agonist in different experimental conditions (t-test, **P < 0.01, ***P < 0.001 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. (D) Ca2+ add-back experiments: after stimulation with BK in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry. 2-APB (50 μM) and SKF96395 (10 μM), nonselective blockers of store-operated Ca2+ channels, significantly inhibited extracellular Ca2+ entry. (E) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (50 μM), or SKF96365. ***P < 0.001 vs. control.
Figure 1.
 
Agonists linked to Gq-coupled receptors activate a store-operated Ca2+ entry. (A) Representative Ca2+ increase after BK (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Experiments similar to these are shown in (A) with endothelin-1 (ET-1, 0.1 μM). (C) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated by each agonist in different experimental conditions (t-test, **P < 0.01, ***P < 0.001 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. (D) Ca2+ add-back experiments: after stimulation with BK in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry. 2-APB (50 μM) and SKF96395 (10 μM), nonselective blockers of store-operated Ca2+ channels, significantly inhibited extracellular Ca2+ entry. (E) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (50 μM), or SKF96365. ***P < 0.001 vs. control.
Figure 2.
 
Store-operated Ca2+ currents activated by BK. (A) In perforated patch experiments, application of BK (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds. Inward currents were measured at −80 mV (mean curve of 10 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) (a, b time points). Curves before (a; baseline) and during (b) BK application are shown. (C) Detection of TRPC channels in cultured bovine TM cells and fresh tissue. Upper: total cell lysate proteins (2–4 μg) were separated by SDS-PAGE and immunoblotted with anti-TRPC1, anti-TRPC4, and anti-TRPC6 (upper left). Results are representative of three experiments. Upper middle: antibodies were previously preincubated with their specific antigen (Ag). Upper right: positive controls were performed by assaying the same antibodies in total rat brain lysate proteins. Lower: similar experiments were performed using protein extracts from fresh bovine TM tissue.
Figure 2.
 
Store-operated Ca2+ currents activated by BK. (A) In perforated patch experiments, application of BK (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds. Inward currents were measured at −80 mV (mean curve of 10 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) (a, b time points). Curves before (a; baseline) and during (b) BK application are shown. (C) Detection of TRPC channels in cultured bovine TM cells and fresh tissue. Upper: total cell lysate proteins (2–4 μg) were separated by SDS-PAGE and immunoblotted with anti-TRPC1, anti-TRPC4, and anti-TRPC6 (upper left). Results are representative of three experiments. Upper middle: antibodies were previously preincubated with their specific antigen (Ag). Upper right: positive controls were performed by assaying the same antibodies in total rat brain lysate proteins. Lower: similar experiments were performed using protein extracts from fresh bovine TM tissue.
Figure 3.
 
Store depletion activates a store-operated Ca2+ entry. (A) Representative Ca2+ increase after TG (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated in each condition (t-test, *P < 0.05, **P < 0.01 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. Ca2+ add-back experiments: after stimulation with TG in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry (C). 2-APB (50 μM; D) and SKF96395 (10 μM; E), nonselective blockers of store-operated Ca2+ channels, significantly blocked the extracellular Ca2+ entry. (F) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (20 μM), or Gd3+ (20 μM). ***P < 0.001 vs. control.
Figure 3.
 
Store depletion activates a store-operated Ca2+ entry. (A) Representative Ca2+ increase after TG (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated in each condition (t-test, *P < 0.05, **P < 0.01 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. Ca2+ add-back experiments: after stimulation with TG in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry (C). 2-APB (50 μM; D) and SKF96395 (10 μM; E), nonselective blockers of store-operated Ca2+ channels, significantly blocked the extracellular Ca2+ entry. (F) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (20 μM), or Gd3+ (20 μM). ***P < 0.001 vs. control.
Figure 4.
 
Store-operated Ca2+ currents activated by store depletion. (A) In whole-cell patch clamp experiments, application of TG (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds Inward currents were measured at −80 mV (mean curve of 6 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) displays a clear inward rectification and very positive reversal potential that supports the presence of CRAC channels. (C) Whole-cell patch clamp experiments with 30 μM IP3 + 10 mM EGTA in the recording pipette. Cell dialysis with IP3 + EGTA activates an extracellular Ca2+ entry that is absent in control experiments in which IP3 was omitted (+0.2 mM EGTA). Same experimental protocol used in (A). Mean curve of six cells (IP3 + EGTA) and five cells (control) is presented. SEM is omitted for clarity. (D) Current-to-voltage relationship for IP3 + EGTA experiments shown in (C) displays a slightly inward rectification with a reversal potential at +19 mV that reflects the combination of CRAC currents together with nonselective cation currents. (E) Effects of different store-operated Ca2+ current blockers on IP3 + EGTA activated currents. Left: current was activated with the same protocol used in (C). After current activation, bath solution was replaced with a DVF. The increase in current magnitude in DVF conditions was blocked by 2-APB (100 μM). **P < 0.01 (baseline vs. DVF or DVF vs. 2-APB). Right: 100 μM La3+ or Gd3+ significantly blocked IP3 + EGTA activated currents. *P < 0.05 (baseline vs. La3+ or Gd3+). (F) Representative experiment using the protocol described in (E, left).
Figure 4.
 
Store-operated Ca2+ currents activated by store depletion. (A) In whole-cell patch clamp experiments, application of TG (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds Inward currents were measured at −80 mV (mean curve of 6 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) displays a clear inward rectification and very positive reversal potential that supports the presence of CRAC channels. (C) Whole-cell patch clamp experiments with 30 μM IP3 + 10 mM EGTA in the recording pipette. Cell dialysis with IP3 + EGTA activates an extracellular Ca2+ entry that is absent in control experiments in which IP3 was omitted (+0.2 mM EGTA). Same experimental protocol used in (A). Mean curve of six cells (IP3 + EGTA) and five cells (control) is presented. SEM is omitted for clarity. (D) Current-to-voltage relationship for IP3 + EGTA experiments shown in (C) displays a slightly inward rectification with a reversal potential at +19 mV that reflects the combination of CRAC currents together with nonselective cation currents. (E) Effects of different store-operated Ca2+ current blockers on IP3 + EGTA activated currents. Left: current was activated with the same protocol used in (C). After current activation, bath solution was replaced with a DVF. The increase in current magnitude in DVF conditions was blocked by 2-APB (100 μM). **P < 0.01 (baseline vs. DVF or DVF vs. 2-APB). Right: 100 μM La3+ or Gd3+ significantly blocked IP3 + EGTA activated currents. *P < 0.05 (baseline vs. La3+ or Gd3+). (F) Representative experiment using the protocol described in (E, left).
Figure 5.
 
Store-operated Ca2+ entry is not necessary for cell contraction, but it modulates the process. (A) Increase in cell contraction after BK (1 μM) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 10). (B) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of BK in control or Ca2+-free solution, after Ca2+ reintroduction or in the presence of 100 μM La3+ (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed. (C) Increase in cell contraction after endothelin-1 (0.1 μM; ET-1) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 6). (D) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of ET-1 in control or Ca2+-free solution and after Ca2+ reintroduction. ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed.
Figure 5.
 
Store-operated Ca2+ entry is not necessary for cell contraction, but it modulates the process. (A) Increase in cell contraction after BK (1 μM) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 10). (B) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of BK in control or Ca2+-free solution, after Ca2+ reintroduction or in the presence of 100 μM La3+ (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed. (C) Increase in cell contraction after endothelin-1 (0.1 μM; ET-1) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 6). (D) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of ET-1 in control or Ca2+-free solution and after Ca2+ reintroduction. ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed.
LlobetA, GasullX, GualA. Understanding trabecular meshwork physiology: a key to the control of intraocular pressure?. News Physiol Sci. 2003;18:205–209. [PubMed]
WeinrebRN, KhawPT. Primary open-angle glaucoma. Lancet. 2004;363:1711–1720. [CrossRef] [PubMed]
WiederholtM, ThiemeH, StumpffF. The regulation of trabecular meshwork and ciliary muscle contractility. Prog Retin Eye Res. 2000;19:271–295. [CrossRef] [PubMed]
Lepple-WienhuesA, RauchR, ClarkAF, GrassmannA, BerweckS, WiederholtM. Electrophysiological properties of cultured human trabecular meshwork cells. Exp Eye Res. 1994;59:305–311. [CrossRef] [PubMed]
WiederholtM, BielkaS, SchweigF, Lutjen-DrecollE, Lepple-WienhuesA. Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye. Exp Eye Res. 1995;61:223–234. [CrossRef] [PubMed]
LlobetA, GualA, PalesJ, BarraquerR, TobiasE, NicolasJM. Bradykinin decreases outflow facility in perfused anterior segments and induces shape changes in passaged BTM cells in vitro. Invest Ophthalmol Vis Sci. 1999;40:113–125. [PubMed]
SotoD, ComesN, FerrerE, et al. Modulation of aqueous humor outflow by ionic mechanisms involved in trabecular meshwork cell volume regulation. Invest Ophthalmol Vis Sci. 2004;45:3650–3661. [CrossRef] [PubMed]
Erickson-LamyK, KorbmacherC, SchumanJS, NathansonJA. Effect of endothelin on outflow facility and accommodation in the monkey eye in vivo. Invest Ophthalmol Vis Sci. 1991;32:492–495. [PubMed]
RaoPV, DengP, SasakiY, EpsteinDL. Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humor outflow facility. Exp Eye Res. 2005;80:197–206. [CrossRef] [PubMed]
Lepple-WienhuesA, StahlF, WiederholtM. Differential smooth muscle-like contractile properties of trabecular meshwork and ciliary muscle. Exp Eye Res. 1991;53:33–38. [CrossRef] [PubMed]
WiederholtM, SchaferR, WagnerU, Lepple-WienhuesA. Contractile response of the isolated trabecular meshwork and ciliary muscle to cholinergic and adrenergic agents. Ger J Ophthalmol. 1996;5:146–153. [PubMed]
ShadeDL, ClarkAF, PangIH. Effects of muscarinic agents on cultured human trabecular meshwork cells. Exp Eye Res. 1996;62:201–210. [CrossRef] [PubMed]
TaoW, PrasannaG, DimitrijevichS, YorioT. Endothelin receptor A is expressed and mediates the [Ca2+]i mobilization of cells in human ciliary smooth muscle, ciliary nonpigmented epithelium, and trabecular meshwork. Curr Eye Res. 1998;17:31–38. [CrossRef] [PubMed]
ParekhAB, PutneyJW, Jr. Store-operated calcium channels. Physiol Rev. 2005;85:757–810. [CrossRef] [PubMed]
BerridgeMJ. Unlocking the secrets of cell signaling. Annu Rev Physiol. 2005;67:1–21. [CrossRef] [PubMed]
ZhangSL, YuY, RoosJ, et al. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature. 2005;437:902–905. [CrossRef] [PubMed]
VigM, PeineltC, BeckA, et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science. 2006;312:1220–1223. [CrossRef] [PubMed]
PrakriyaM, FeskeS, GwackY, SrikanthS, RaoA, HoganPG. Orai1 is an essential pore subunit of the CRAC channel. Nature. 2006;443:230–233. [CrossRef] [PubMed]
YerominAV, ZhangSL, JiangW, YuY, SafrinaO, CahalanMD. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature. 2006;443:226–229. [CrossRef] [PubMed]
HuangGN, ZengW, KimJY, et al. STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels. Nat Cell Biol. 2006;8:1003–1010. [CrossRef] [PubMed]
RamseyIS, DellingM, ClaphamDE. An introduction to TRP channels. Annu Rev Physiol. 2006;68:619–647. [CrossRef] [PubMed]
AmbudkarIS. Ca2+ signaling microdomains: platforms for the assembly and regulation of TRPC channels. Trends Pharmacol Sci. 2006;27:25–32. [CrossRef] [PubMed]
MehtaD, AhmmedGU, PariaBC, et al. RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry: role in signaling increased endothelial permeability. J Biol Chem. 2003;278:33492–33500. [CrossRef] [PubMed]
MehtaD, MalikAB. Signaling mechanisms regulating endothelial permeability. Physiol Rev. 2006;86:279–367. [CrossRef] [PubMed]
SinghI, KnezevicN, AhmmedGU, KiniV, MalikAB, MehtaD. Galpha q-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin. J Biol Chem. 2007;282:7833–7843. [CrossRef] [PubMed]
StamerWD, SeftorRE, WilliamsSK, SamahaHA, SnyderRW. Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion. Curr Eye Res. 1995;14:611–617. [CrossRef] [PubMed]
GasullX, FerrerE, LlobetA, et al. Cell membrane stretch modulates the high-conductance Ca2+ -activated K+ channel in bovine trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2003;44:706–714. [CrossRef] [PubMed]
GavaraN, SunyerR, Roca-CusachsP, FarreR, RotgerM, NavajasD. Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy. J Appl Physiol. 2006;101:512–520. [CrossRef] [PubMed]
PelhamRJ, Jr, WangY. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA. 1997;94:13661–13665. [CrossRef] [PubMed]
KarlMO, FleischhauerJC, StamerWD, et al. Differential P1-purinergic modulation of human Schlemm’s canal inner-wall cells. Am J Physiol Cell Physiol. 2005;288:C784–C794. [PubMed]
Lepple-WienhuesA, StahlF, WillnerU. Endothelin-evoked contractions in bovine ciliary muscle and trabecular meshwork: interaction with calcium, nifedipine and nickel. Curr Eye Res. 1991;10:983–989. [CrossRef] [PubMed]
KohmotoH, MatsumotoS, SerizawaT. Effects of endothelin-1 on [Ca2+]i and pHi in trabecular meshwork cells. Curr Eye Res. 1994;13:197–202. [CrossRef] [PubMed]
Lepple-WienhuesA, StahlF, WunderlingD, WiederholtM. Effects of endothelin and calcium channel blockers on membrane voltage and intracellular calcium in cultured bovine trabecular meshwork cells. Ger J Ophthalmol. 1992;1:159–163. [PubMed]
SotoD, PintorJ, PeralA, GualA, GasullX. Effects of dinucleoside polyphosphates on trabecular meshwork cells and aqueous humor outflow facility. J Pharmacol Exp Ther. 2005;314:1042–1051. [CrossRef] [PubMed]
ZhouH, IwasakiH, NakamuraT, et al. 2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry. Biochem Biophys Res Commun. 2007;352:277–282. [CrossRef] [PubMed]
MerrittJE, ArmstrongWP, BenhamCD, et al. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J. 1990;271:515–522. [PubMed]
XuSZ, ZengF, BoulayG, GrimmC, HarteneckC, BeechDJ. Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect. Br J Pharmacol. 2005;145:405–414. [PubMed]
HoganPG, RaoA. Dissecting ICRAC, a store-operated calcium current. Trends Biochem Sci. 2007;32:235–245. [CrossRef] [PubMed]
FierroL, ParekhAB. On the characterisation of the mechanism underlying passive activation of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia cells. J Physiol. 1999;520 Pt 2:407–416. [PubMed]
ParekhAB. Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane. J Physiol. 2003;547:333–348. [CrossRef] [PubMed]
BakowskiD, ParekhAB. Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes. Cell Calcium. 2002;32:379–391. [CrossRef] [PubMed]
ThiemeH, SchimmatC, MunzerG, et al. Endothelin antagonism: effects of FP receptor agonists prostaglandin F2alpha and fluprostenol on trabecular meshwork contractility. Invest Ophthalmol Vis Sci. 2006;47:938–945. [CrossRef] [PubMed]
RosenthalR, ChoritzL, SchlottS, et al. Effects of ML-7 and Y-27632 on carbachol- and endothelin-1-induced contraction of bovine trabecular meshwork. Exp Eye Res. 2005;80:837–845. [CrossRef] [PubMed]
ParekhAB. Functional consequences of activating store-operated CRAC channels. Cell Calcium. 2007;42:111–121. [CrossRef] [PubMed]
ShimuraM, YasudaK, NakzawaT, KashiwagiK. The effect of unoprostone isopropyl on Ca2+ release-activated Ca2+ currents in cultured monkey trabecular meshwork cells and ciliary muscle cells. J Ocul Pharmacol Ther. 2006;22:219–226. [CrossRef] [PubMed]
SoboloffJ, SpassovaMA, TangXD, HewavitharanaT, XuW, GillDL. Orai1 and STIM reconstitute store-operated calcium channel function. J Biol Chem. 2006;281:20661–20665. [CrossRef] [PubMed]
SmythJT, DehavenWI, JonesBF, et al. Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP. Biochim Biophys Acta. 2006;1763:1147–1160. [CrossRef] [PubMed]
AmbudkarIS. TRPC1: a core component of store-operated calcium channels. Biochem Soc Trans. 2007;35:96–100. [CrossRef] [PubMed]
OngHL, ChengKT, LiuX, et al. Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx: evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem. 2007;282:9105–9116. [CrossRef] [PubMed]
Peppiatt-WildmanCM, AlbertAP, SalehSN, LargeWA. Endothelin-1 activates a Ca2+-permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes. J Physiol. 2007;580:755–764. [CrossRef] [PubMed]
PariaBC, BairAM, XueJ, YuY, MalikAB, TiruppathiC. Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-kappaB activation in endothelial cells. J Biol Chem. 2006;281:20715–20727. [CrossRef] [PubMed]
YuanJP, ZengW, HuangGN, WorleyPF, MuallemS. STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels. Nat Cell Biol. 2007;9:636–645. [CrossRef] [PubMed]
WoldeMussieE, RuizG. Effect of histamine on signal transduction in cultured human trabecular meshwork cells. Curr Eye Res. 1992;11:987–995. [CrossRef] [PubMed]
TaoL, HarrisAL. 2-Aminoethoxydiphenyl borate directly inhibits channels composed of connexin26 and/or connexin32. Mol Pharmacol. 2007;71:570–579. [PubMed]
BerridgeMJ, BootmanMD, RoderickHL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–529. [PubMed]
CooperDM, KarpenJW, FaganKA, MonsNE. Ca(2+)-sensitive adenylyl cyclases. Adv Second Messenger Phosphoprotein Res. 1998;32:23–51. [PubMed]
LinS, FaganKA, LiKX, ShaulPW, CooperDM, RodmanDM. Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry. J Biol Chem. 2000;275:17979–17985. [CrossRef] [PubMed]
ChangWC, NelsonC, ParekhAB. Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells. FASEB J. 2006;20:2381–2383. [CrossRef] [PubMed]
DolmetschRE, XuK, LewisRS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature. 1998;392:933–936. [CrossRef] [PubMed]
ThiemeH, NuskovskiM, NassJU, PleyerU, StraussO, WiederholtM. Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A. Invest Ophthalmol Vis Sci. 2000;41:4240–4246. [PubMed]
RaoPV, DengPF, KumarJ, EpsteinDL. Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632. Invest Ophthalmol Vis Sci. 2001;42:1029–1037. [PubMed]
TianB, KaufmanPL. Effects of the Rho kinase inhibitor Y-27632 and the phosphatase inhibitor calyculin A on outflow facility in monkeys. Exp Eye Res. 2005;80:215–225. [CrossRef] [PubMed]
ZhangW, MengH, LiZH, ShuZ, MaX, ZhangBX. Regulation of STIM1, store-operated Ca2+ influx, and nitric oxide generation by retinoic acid in rat mesangial cells. Am J Physiol Renal Physiol. 2007;292:F1054–F1064. [PubMed]
KeeC, KaufmanPL, GabeltBT. Effect of 8-Br cGMP on aqueous humor dynamics in monkeys. Invest Ophthalmol Vis Sci. 1994;35:2769–2773. [PubMed]
StumpffF, StraussO, BoxbergerM, WiederholtM. Characterization of maxi-K-channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate. Invest Ophthalmol Vis Sci. 1997;38:1883–1892. [PubMed]
WiederholtM, SturmA, Lepple-WienhuesA. Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci. 1994;35:2515–2520. [PubMed]
GilabertR, GasullX, PalesJ, BelmonteC, BergaminiMV, GualA. Facility changes mediated by cAMP in the bovine anterior segment in vitro. Vision Res. 1997;37:9–15. [CrossRef] [PubMed]
PolanskyJR, KurtzRM, AlvaradoJA, WeinrebRN, MitchellMD. Eicosanoid production and glucocorticoid regulatory mechanisms in cultured human trabecular meshwork cells. Prog Clin Biol Res. 1989;312:113–138. [PubMed]
WeinrebRN, MitchellMD. Prostaglandin production by cultured cynomolgus monkey trabecular meshwork cells. Prostaglandins Leukot Essent Fatty Acids. 1989;36:97–100. [CrossRef] [PubMed]
WebbJG, ShearerTW, YatesPW, MukhinYV, CrossonCE. Bradykinin enhancement of PGE2 signalling in bovine trabecular meshwork cells. Exp Eye Res. 2003;76:283–289. [CrossRef] [PubMed]
WebbJG, HusainS, YatesPW, CrossonCE. Kinin modulation of conventional outflow facility in the bovine eye. J Ocul Pharmacol Ther. 2006;22:310–316. [CrossRef] [PubMed]
StumpffF, BoxbergerM, KraussA, et al. Stimulation of cannabinoid (CB1) and prostanoid (EP2) receptors opens BKCa channels and relaxes ocular trabecular meshwork. Exp Eye Res. 2005;80:697–708. [CrossRef] [PubMed]
ShengJZ, BraunAP. Small and intermediate conductance, Ca-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells. Am J Physiol Cell Physiol. 2007;293:C458–C467. [CrossRef] [PubMed]
Figure 1.
 
Agonists linked to Gq-coupled receptors activate a store-operated Ca2+ entry. (A) Representative Ca2+ increase after BK (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Experiments similar to these are shown in (A) with endothelin-1 (ET-1, 0.1 μM). (C) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated by each agonist in different experimental conditions (t-test, **P < 0.01, ***P < 0.001 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. (D) Ca2+ add-back experiments: after stimulation with BK in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry. 2-APB (50 μM) and SKF96395 (10 μM), nonselective blockers of store-operated Ca2+ channels, significantly inhibited extracellular Ca2+ entry. (E) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (50 μM), or SKF96365. ***P < 0.001 vs. control.
Figure 1.
 
Agonists linked to Gq-coupled receptors activate a store-operated Ca2+ entry. (A) Representative Ca2+ increase after BK (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Experiments similar to these are shown in (A) with endothelin-1 (ET-1, 0.1 μM). (C) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated by each agonist in different experimental conditions (t-test, **P < 0.01, ***P < 0.001 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. (D) Ca2+ add-back experiments: after stimulation with BK in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry. 2-APB (50 μM) and SKF96395 (10 μM), nonselective blockers of store-operated Ca2+ channels, significantly inhibited extracellular Ca2+ entry. (E) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (50 μM), or SKF96365. ***P < 0.001 vs. control.
Figure 2.
 
Store-operated Ca2+ currents activated by BK. (A) In perforated patch experiments, application of BK (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds. Inward currents were measured at −80 mV (mean curve of 10 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) (a, b time points). Curves before (a; baseline) and during (b) BK application are shown. (C) Detection of TRPC channels in cultured bovine TM cells and fresh tissue. Upper: total cell lysate proteins (2–4 μg) were separated by SDS-PAGE and immunoblotted with anti-TRPC1, anti-TRPC4, and anti-TRPC6 (upper left). Results are representative of three experiments. Upper middle: antibodies were previously preincubated with their specific antigen (Ag). Upper right: positive controls were performed by assaying the same antibodies in total rat brain lysate proteins. Lower: similar experiments were performed using protein extracts from fresh bovine TM tissue.
Figure 2.
 
Store-operated Ca2+ currents activated by BK. (A) In perforated patch experiments, application of BK (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds. Inward currents were measured at −80 mV (mean curve of 10 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) (a, b time points). Curves before (a; baseline) and during (b) BK application are shown. (C) Detection of TRPC channels in cultured bovine TM cells and fresh tissue. Upper: total cell lysate proteins (2–4 μg) were separated by SDS-PAGE and immunoblotted with anti-TRPC1, anti-TRPC4, and anti-TRPC6 (upper left). Results are representative of three experiments. Upper middle: antibodies were previously preincubated with their specific antigen (Ag). Upper right: positive controls were performed by assaying the same antibodies in total rat brain lysate proteins. Lower: similar experiments were performed using protein extracts from fresh bovine TM tissue.
Figure 3.
 
Store depletion activates a store-operated Ca2+ entry. (A) Representative Ca2+ increase after TG (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated in each condition (t-test, *P < 0.05, **P < 0.01 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. Ca2+ add-back experiments: after stimulation with TG in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry (C). 2-APB (50 μM; D) and SKF96395 (10 μM; E), nonselective blockers of store-operated Ca2+ channels, significantly blocked the extracellular Ca2+ entry. (F) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (20 μM), or Gd3+ (20 μM). ***P < 0.001 vs. control.
Figure 3.
 
Store depletion activates a store-operated Ca2+ entry. (A) Representative Ca2+ increase after TG (1 μM) application in a TM cell in control solution (1.3 mM Ca2+). In Ca2+-free conditions or in the presence of 2-APB (50 μM), the sustained phase of the Ca2+ transient was greatly reduced. (B) Time to recover the 70% of the Ca2+ increase as a measurement of the sustained Ca2+ entry activated in each condition (t-test, *P < 0.05, **P < 0.01 control vs. free Ca2+ or 2-APB). Number of analyzed cells is shown inside the columns. Ca2+ add-back experiments: after stimulation with TG in Ca2+-free bath, Ca2+ was reintroduced to measure store-activated Ca2+ entry (C). 2-APB (50 μM; D) and SKF96395 (10 μM; E), nonselective blockers of store-operated Ca2+ channels, significantly blocked the extracellular Ca2+ entry. (F) Extracellular Ca2+ entry in control conditions compared with the increase in the presence of 2-APB, La3+ (20 μM), or Gd3+ (20 μM). ***P < 0.001 vs. control.
Figure 4.
 
Store-operated Ca2+ currents activated by store depletion. (A) In whole-cell patch clamp experiments, application of TG (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds Inward currents were measured at −80 mV (mean curve of 6 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) displays a clear inward rectification and very positive reversal potential that supports the presence of CRAC channels. (C) Whole-cell patch clamp experiments with 30 μM IP3 + 10 mM EGTA in the recording pipette. Cell dialysis with IP3 + EGTA activates an extracellular Ca2+ entry that is absent in control experiments in which IP3 was omitted (+0.2 mM EGTA). Same experimental protocol used in (A). Mean curve of six cells (IP3 + EGTA) and five cells (control) is presented. SEM is omitted for clarity. (D) Current-to-voltage relationship for IP3 + EGTA experiments shown in (C) displays a slightly inward rectification with a reversal potential at +19 mV that reflects the combination of CRAC currents together with nonselective cation currents. (E) Effects of different store-operated Ca2+ current blockers on IP3 + EGTA activated currents. Left: current was activated with the same protocol used in (C). After current activation, bath solution was replaced with a DVF. The increase in current magnitude in DVF conditions was blocked by 2-APB (100 μM). **P < 0.01 (baseline vs. DVF or DVF vs. 2-APB). Right: 100 μM La3+ or Gd3+ significantly blocked IP3 + EGTA activated currents. *P < 0.05 (baseline vs. La3+ or Gd3+). (F) Representative experiment using the protocol described in (E, left).
Figure 4.
 
Store-operated Ca2+ currents activated by store depletion. (A) In whole-cell patch clamp experiments, application of TG (1 μM) progressively activated extracellular Ca2+ entry. Cells were clamped at 0 mV, and 500-ms ramps from −100 to + 100 mV were applied every 5 seconds Inward currents were measured at −80 mV (mean curve of 6 cells is presented; SEM is omitted for clarity). (B) Current-to-voltage relationship for the experiments shown in (A) displays a clear inward rectification and very positive reversal potential that supports the presence of CRAC channels. (C) Whole-cell patch clamp experiments with 30 μM IP3 + 10 mM EGTA in the recording pipette. Cell dialysis with IP3 + EGTA activates an extracellular Ca2+ entry that is absent in control experiments in which IP3 was omitted (+0.2 mM EGTA). Same experimental protocol used in (A). Mean curve of six cells (IP3 + EGTA) and five cells (control) is presented. SEM is omitted for clarity. (D) Current-to-voltage relationship for IP3 + EGTA experiments shown in (C) displays a slightly inward rectification with a reversal potential at +19 mV that reflects the combination of CRAC currents together with nonselective cation currents. (E) Effects of different store-operated Ca2+ current blockers on IP3 + EGTA activated currents. Left: current was activated with the same protocol used in (C). After current activation, bath solution was replaced with a DVF. The increase in current magnitude in DVF conditions was blocked by 2-APB (100 μM). **P < 0.01 (baseline vs. DVF or DVF vs. 2-APB). Right: 100 μM La3+ or Gd3+ significantly blocked IP3 + EGTA activated currents. *P < 0.05 (baseline vs. La3+ or Gd3+). (F) Representative experiment using the protocol described in (E, left).
Figure 5.
 
Store-operated Ca2+ entry is not necessary for cell contraction, but it modulates the process. (A) Increase in cell contraction after BK (1 μM) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 10). (B) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of BK in control or Ca2+-free solution, after Ca2+ reintroduction or in the presence of 100 μM La3+ (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed. (C) Increase in cell contraction after endothelin-1 (0.1 μM; ET-1) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 6). (D) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of ET-1 in control or Ca2+-free solution and after Ca2+ reintroduction. ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed.
Figure 5.
 
Store-operated Ca2+ entry is not necessary for cell contraction, but it modulates the process. (A) Increase in cell contraction after BK (1 μM) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 10). (B) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of BK in control or Ca2+-free solution, after Ca2+ reintroduction or in the presence of 100 μM La3+ (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed. (C) Increase in cell contraction after endothelin-1 (0.1 μM; ET-1) application in physiological solution (control medium; n = 7) or in Ca2+-free medium (n = 6). (D) Summary of effects on cell contraction: initial baseline is compared with the maximal contraction exerted in the presence of ET-1 in control or Ca2+-free solution and after Ca2+ reintroduction. ***P < 0.001 (baseline vs. each condition). Nonsignificant (ns) comparisons are also displayed.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×