35 research outputs found
Measurement of intracellular Ca<sup>2+</sup>, [Ca<sup>2+</sup>]<sub>i</sub>, levels showing strong cross-talk between TRPV4 and the KCa channels in mCCDcl1.
<p>All traces are representative examples as indicated. Addition of GSK101 or the blockers is indicated by the length of the “bar” below each symbol in the individual tracings. <b>A.</b> In the absence of TRPV4 stimulation, [Ca<sup>2+</sup>]<sub>i</sub> levels remain relative low in all cells. <b>B.</b> Upon activation of TPRV4 with the selective agonist, GSK101, [Ca<sup>2+</sup>]<sub>i</sub> displays a biphasic response, rising to a peak [Ca<sup>2+</sup>]<sub>i</sub> level with the first few minutes, then partial relaxes to a steady-state plateau which is above the basal [Ca<sup>2+</sup>]<sub>i</sub> levels. <b>C.</b> The average changes in [Ca<sup>2+</sup>]<sub>i</sub> are given showing the basal (GSK-) and peak (GSK+) [Ca<sup>2+</sup>]<sub>i</sub> levels upon stimulation with GSK101 (3 nM). <b>D.</b> Effect of addition of a cocktail of KCa channel inhibitors (Apamin, 300 nM, IbTX, 100 nM, TRAM-34, 300 nM, and TQ, 5 nM) on [Ca<sup>2+</sup>]<sub>i</sub> prior to activation of TRPV4 with GSK101. No response is apparent. <b>E.</b> Effect of addition of the same cocktail of KCa blockers as in panel <b>D</b>, but added after GSK101 activation of TRPV4. The cocktail of blockers markedly depressed the TRPV4-mediated [Ca<sup>2+</sup>]<sub>i</sub> levels. <b>F.</b> Summary of the average decrease in [Ca<sup>2+</sup>]<sub>i</sub> upon addition of the cocktail of KCa blockers (4 Blockers) before after activation of TRPV4. Addition of the 4 Blockers had not affect in the absence of GSK101 (-), averaging 0.4 ± 2.5 nM (n = 24). In the presence of GSK101 (+), the 4 Blockers induce a marked decrease in [Ca<sup>2+</sup>]<sub>i</sub> of 477 ± 45 nM (n = 24, P<0.001) with the 4 Blockers, demonstrates a strong functional cross-talk between TRPV4, [Ca<sup>2+</sup>]<sub>I</sub>, and KCa channels. <b>G.</b> Effect of selective inhibition of BK (IbTX, 100 nM) or ROMK (TQ, 5 nM) on GSK101-induced [Ca<sup>2+</sup>]<sub>i</sub> levels showing a significant reduction in [Ca<sup>2+</sup>]<sub>i</sub> with either blocker. <b>H.</b> Effect of selective inhibition of IK1 (TRAM-34, 300 nM) or SKs (Apamin, 300 nM) on GSK101-induced [Ca<sup>2+</sup>]<sub>i</sub> levels showing a significant reduction in [Ca<sup>2+</sup>]<sub>i</sub> with either blocker. <b>I.</b> Summary results of the actions of each of the KCa blockers on GSK101-induced [Ca<sup>2+</sup>]<sub>i</sub> levels showing a pronounced decrease in [Ca<sup>2+</sup>]<sub>i</sub> levels with each of the blockers on TRPV4-mediated Ca2+ influx. The reduction in [Ca<sup>2+</sup>]<sub>i</sub> averaged 236 ± 45 nM (n = 24) for IbTX, 175 ± 18 nM (n = 24) for TQ, 193 ± 22 nM (n = 24) for TRAM-34, and 208 ± 14 nM (n = 24) for Apamin (P<0.001 for each case). The individual responses do not differ among blockers. In contrast, the effect of the individual blockers is significantly less than that observed for the 4 Blocker combination in 8F (P<0.01).</p
Patch clamp analysis showing membrane hyperpolarization following TRPV4-mediated Ca2+ influx and the effect of KCa channel blockers in mCCDcl1 cells grown on glass coverslips.
<p>Current clamp recordings were initiated in ECS with 2 mM CaCl<sub>2</sub> and 140 nM NaCl (normal ECS) with no current injection. The membrane potential (Vm) recorded at the beginning of each cell measurement is marked by a horizontal arrow. After a stable Vm was achieved, the normal ECS was quickly changed to the ECS without 2 mM Ca<sup>2+</sup> (nominally Ca<sup>2+</sup> free ECS, arrow 1), but still with 140 mM NaCl. A slow drift of the Vm (<10 mV) occurred, initially, but stabilized within 1 min. Whereupon, 10 nM GSK101 dissolved in a nominally Ca<sup>2+</sup>-free ECS was applied to the cell (arrow 2) which led to a depolarization of Vm, due to TRPV4-mediated Na<sup>+</sup> influx into the cell. Once the depolarization reached a new plateau (between -10 mV to 5 mV), the perfusion line was switched to the one with 10 nM GSK101 dissolved in normal ECS (with Ca<sup>2+</sup>, arrow 3), which induced rapid Ca<sup>2+</sup> influx, and was continued for at least 3 min. The “instant” and significant Ca<sup>2+</sup> increase in the cytoplasm immediately activated KCa channels which elicited an instant and strong hyperpolarization (20–30 mV) within 2–3 sec (first hyperpolarization peak). This was followed by a slow further hyperpolarization of Vm in normal ECS. If KCa blockers were applied immediately after the first hyperpolarization peak, the blockers lead to a marked reduction in the extent of the Vm hyperpolarization as shown for all KCa blockers (<b>B-E</b>). <b>A.</b> Representative Vm tracing in a control cell showing that rapid induction of Ca<sup>2+</sup> influx (arrow 3) lead to a rapid hyperpolarization of Vm within 2–3 sec (first hyperpolarization peak) followed by a second slower hyperpolarization over the remaining 3 min. <b>B-E.</b> Representative tracing showing the effect of addition of KCa channel blockers (arrows 4–7) after the first hyperpolarization peak which lead to marked reduction of hyperpolarization response over the subsequent 3-min perfusion period following addition of IbTX (<b>B</b>), TRAM-34 (<b>C</b>), apamin (<b>D</b>), or all three blockers (<b>E</b>). <b>F.</b> Summary of the blocking effects of KCa blockers where the decrease in the hyperpolarization of Vm was normalized to first hyperpolarization peak response for each cell showing a highly significant reduction in the hyperpolarization response, averaging 53.4 ± 11% for IbTX, 48.4 ± 11.7% for TRAM-34, 44.4 ± 9.7% for Apamin, and 42.1 ± 10.4% for the 3-blockers, **P<0.01 (n’s are given in the bar graph for each blocker).</p
Immunocytochemistry fluorescence images showing results of staining for ion channels in mouse kidney cortical sections at low magnification.
<p>Results show staining for the ion channel of interest and for AQP2 (Red, 594 nM), a marker of CNT and CCD in the cortex. A bifurcation of AQP2-positive tubules was used to identify CNT segments (above the bifurcation) and CCD segments (single tubule below the bifurcation). <b>A.</b> SK1 staining of tubule segments (green, 488 nm) shows strong staining of many cells along both the CNT and CCD (SK1, labels CNT and CCD). Merged images with AQP2 staining verify the CNT and CCD sites of localization. <b>B.</b> SK3 staining of tubule segments (green, 488 nm) shows prominent staining of most cells along both the CNT and CCD (SK3, labels CNT and CCD). Merged images with AQP2 verify the CNT and CCD sites of localization. <b>C.</b> IK1 staining (red, 594 nm) is strong in most cells of both the CNT and CCD (IK1, labels CNT and CCD) as verified in the Merged image. <b>D.</b> BKα staining (green, 488 nm) is prominent in most cells of both the CCD (BKα, CCD) and along the CNT (data not shown) as verified in the Merged images. <b>E.</b> TRPV4 staining (green, 488 nm) is apparent in most cells of the CCD (TRPV4, CCD) and in the CNT (data not shown) as verified in the Merged images. All fluorescence images were obtained at 40x (oil) with a Zeiss Axioskop 40 microscope using a AxioCam MRm CCD camera.</p
Differential expression of ion channels between principal cells (PC) and intercalated cells (IC) in the mouse kidney CCD.
<p>AQP2 staining was used to identify PC, the AQP2-positive cells, and IC, the APQ2-negative cells. Bar graphs give the mean ± SEM for the normalized intensities for each KCa channel for both PC and IC. The number of PC and IC cells analyzed, n, is indicated on the bar graphs. The images of CCD show representative immunofluorescence examples for each KCa channel and AQP2 (40X) where one or two PC (AQP2-positive) and IC (AQP2-negative) cells are labelled as “PC” or “IC.” The outer border of the tubule in each image is indicated by the dashed white line (indicating the basal side or anti-luminal side of tubular cells). “L” identifies the tubular lumen. The immunofluorescence intensity was determined for each KCa channel using ImageJ and normalized to the intensity levels of AQP2 expression in PC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155006#sec002" target="_blank">Methods</a>). Each example gives staining for AQP2 (red), the KCa channel (green), and the merged image (AQP2, KCa channel) that includes DAPI staining to identify nuclei. <b>A.</b> Expression of SK1 in PC and IC showing dominant relative expression in IC over PC (P<0.001). SK1 expression was apparent within the cytosol and along the luminal border of both PC and IC as apparent in the representative images. Light staining along the basolateral border is also apparent in some cells. <b>B.</b> Expression of SK3 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). SK3 expression was apparent within the cytosol with light expression along the basal aspect of some cells and more dominant expression along the luminal border of PC. <b>C.</b> Expression of IK1 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). IK1 expression was apparent within the cytosol, especially of IC, but also apparent along the luminal border of PC with light staining along the basal aspect of the cells. <b>D.</b> Expression of BKα in PC and IC showing strong staining along the luminal border of both PC and IC. However, the intensity of staining between PC and IC was found to vary depending on the primary antibody used for the immunostaining. In our hands, the Alomone APC-151 BKα antibody staining was most apparent in PC over IC (P<0.001, compare bar graph labeled APC-151 with APC-107). In contrast, the Alomone APC-107 BKα antibody staining was most apparent in IC over PC. Since the antibodies were made to different epitopes of the BKα channel, such variations can be anticipated (see text).</p
Differential mRNA expression levels of ion channels in mouse kidney.
<p><b>A.</b> qPCR anlysis of mRNA expression levels of marker channels relative to the AQP2 water channel. As in mCCDcl1 cells, the mechanosensitive TRPV4 channel mRNA levels and ROMK mRNA levels are relatively high while mRNA levels for a second mechanosensitve TRP channel, TRPV2, are very low. For comparison mRNA expression levels for all channels are also given, relative to AQP2 mRNA levels, in the accompanying <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155006#pone.0155006.t001" target="_blank">Table 1</a>. <b>B.</b> qPCR analysis of mRNA expression levels of KCa channels in mouse kidney relative to the ROMK channel. It shows that SK1, SK2, and SK3 are expressed at relative high mRNA levels while IK1 and BKα are expressed at moderate mRNA levels. <b>C.</b> mRNA expression of BK channel subunits relative to BKα mRNA levels. The results show high relative mRNA levels for BKβ4, moderate mRNA levels for BKβ2, and low mRNA levels for BKβ4.</p
Differential mRNA and protein expression levels of ion channels in mCCDcl1 cells.
<p><b>A.</b> qPCR analysis of mRNA expression levels of marker channels relative to the AQP2 water channel. The mechanosensitive TRPV4 channel mRNA levels and ROMK mRNA levels are relatively high while a second mechanosensitive TRP channel, TRPV2, are very low. For comparison, mRNA expression levels for all channels are also given, relative to AQP2 mRNA levels, in the accompanying <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155006#pone.0155006.t001" target="_blank">Table 1</a>. <b>B.</b> qPCR mRNA expression levels of KCa channels relative to the ROMK channel. It shows that SK1, SK3, IK1 are expressed at relative high mRNA levels, BKα at moderate mRNA levels, and SK2 at low mRNA levels. <b>C.</b> mRNA expression of BK channel subunits relative to BKα mRNA levels. It shows high relative mRNA levels for BKβ1 and BKβ2, but relatively low levels for BKβ4. <b>D.</b> Immunoblots of mCCDcl1 cells for the key channels showing appropriate protein bands for TRPV4 (98 kD), SK1 (64 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD).</p
The effect of TRPV4 activation and KCa channel blockers on transepithelial electrical resistance (TEER) of mCCDcl1 cells grown to confluency on permeable supports (see Materials and Methods).
<p>Addition of GSK101 or the blockers is indicated by the length of the “bar” below each symbol in the individual tracings. <b>A.</b> Representative time course of TEER measurements under basal conditions showing stable TEER values over the time course of the experiments. <b>B.</b> Representative example of the effect of adding GSK101 to the outside basolateral compartment (Basolateral GSK) or to the luminal (apical) compartment (Luminal GSK). GSK101 had no apparent affect when added to the basolateral media, but showed a consistent decrease in TEER (reflecting activation of TRPV4 and other channels) when added to the luminal compartment. <b>C.</b> Example showing the effect of addition of the cocktail of KCa blockers (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155006#pone.0155006.g008" target="_blank">Fig 8</a>) on the GSK101-induced reduction in TEER. Addition of the blockers lead to an increase in TEER levels, results consistent with inhibition of [Ca<sup>2+</sup>]<sub>i</sub> activated KCa channels. <b>D.</b> Representative example showing the effect of SK inhibition (Apamin) on the GSK101-activated TEER values. <b>E.</b> Representative example showing the effect of BK inhibition (IbTX) on the GSK101-activated TEER values. <b>F.</b> Representative example showing the effect of IK1 inhibition (TRAM-34) on the GSK101-activated TEER values. <b>G.</b> Representative example showing the effect of ROMK inhibition (TQ) on the GSK101-activated TEER values. <b>H.</b> Summary results showing the effect of GSK101 activation of TRPV4 on TEER values (GSK, luminal compartment) and the subsequent individual KCa channel blockers (Luminal Blockers) on the TRPV4-activated TEER measurements. Each blocker (TRAM-34, Apamin, IbTX, TQ) or a cocktail (4X Blockers) of KCa channel blockers showed an increase in TEER. The decrease in TEER upon addition of GSK101 averaged 0.74 ± 0.027 (n = 3). The average increase in the relative TEER for the blockers in the presence of GSK101 was 0.17 ± 0.03 for the 4 Blockers, 0.08 ± 0.02 (n = 3) for TRAM-34, 0.09 ± 0.02 (n = 3) for Apamin, 0.09 ± 0.02 (n = 3) for IbTX, and 0.09 ± 0.02 (n = 3) TQ. Although the response to the 4 Blockers appears much larger, statistically there was no significant difference in the responses among the blocker sets.</p