Figure 1.
Expression and distribution of Ca (V) 1.2 and 1.3 channels in mouse lens.
A and B. RT-PCR-based confirmation of expression of α1C (Ca (V) 1.2), α1D (Ca (V) 1.3), α2δ and β2 (auxiliary subunit genes) in P2 (lane 1) and P21 (lane 2) in mouse lenses. As shown in Panel A, the expression of Ca (V) 1.1 and 1.4 expression was found to be minimal relative to the expression profile of Ca (V) 1.2 and 1.3 in the same mouse lenses, under identical RT-PCR conditions. A –RT control was used in the RT-PCR experiments. B. Immunoblotting-based confirmation of the presence of Ca (V) 1.2 and 1.3 channel proteins in P21 mouse lens membrane enriched [Lanes 2 (75 µg protein) and 3 (125 µg protein)] fractions. The soluble lens fraction (Lane 1, 100 µg protein) did not exhibit immunopositive reactivity against the same antibodies. Arrows indicate the native Ca (V) 1.2 and 1.3 proteins. C. Distribution of Ca (V) 1.2 in P21 mouse lens equatorial sections based on immunofluorescence analysis. Right panel shows a magnified area (boxed in the left panel) and reveals the discretely clustered organization of Ca (V) 1.2 (arrows) at the short arms of the lens hexagonal fiber cell. D. Distribution analysis of Ca (V) 1.3 in P21 mouse lens equatorial sections reveals its intense localization to the short arm (right panel) of the hexagonal lens fiber cell. Bars: 20 µm. Epi: Epithelium.
Figure 2.
Felodipine and Nifedipine-induced lens opacification in ex-vivo cultured mouse lenses.
A. Treatment of organ cultured (P21 to P26) mouse lenses with felodipine (10 µM) induced progressive lens opacity (subcapsular to cortical) with increasing time. Control lenses treated with DMSO (0.025%) remained transparent throughout the experiment. B. The felodipine treated lenses showed significant (* P<0.01) increase in protein secretion/leakage (µg/ml media/lens) into the media as compared to control lenses, based on the mean value from an n = 4 to 6 samples. D. Mouse lenses (P21 to P26) treated with nifedipine (25 µM) exhibited cortical and nuclear opacity after 48 hr of drug treatment, while control lenses treated with DMSO alone (0.025%) remained transparent. C. Both felodipine and nifedipine treated mouse lenses showed a significant (* P<0.01; n = 4–6) decrease in lens wet weight (mg/lens) compared to the corresponding controls.
Figure 3.
Inhibition of LTCCs by felodipine induces extensive disorganization and swelling of cortical fiber cells in ex-vivo organ cultured mouse lenses without any noticeable cell death.
Treatment of organ cultured mouse lenses (P21 to P26) with felodipine (10 µM) induced progressive disorganization of cortical fiber cells (arrows) starting from 6 hrs of treatment. After 24 hrs of drug treatment, lens cortical fibers exhibited extensive swelling (see inserts and arrows) with large intracellular spaces based on light microscope examination (H&E staining) (A) and fluorescence microscopy analysis of equatorial plane tissue specimens using wheat germ agglutinin staining (B & C). The images in Panel C indicate the magnified area depicted in Panel B with squares. DMSO treated control lenses remained histologically intact at both 6 (not shown) and 24 hrs of incubation. D. TUNEL and Hoechst staining of felodipine treated (both 6 and 24 hr) and control lenses reveals comparable staining patterns for both the TUNEL positive (green cells indicated with arrows) and cell nuclei (in blue). Bars indicate magnification. Epi: Epithelium, CF: Cortical fibers, NF: Nuclear fibers.
Figure 4.
Felodipine-induced changes in protein profile of ex-vivo organ cultured mouse lenses.
Felodipine (10 µM for 6 hrs or 24 hrs) and the corresponding DMSO treated control mouse lenses (p21 to P26) were homogenized and separated into soluble (100,000×g supernatant) and insoluble (membrane-enriched) fractions. Equal amounts of protein from soluble (A, C) and insoluble (B, D) fractions were then separated by gradient SDS-PAGE (4–20%) and stained with GelCode blue. Protein bands that exhibited obvious differences in staining intensity between the felodipine treated and control lenses (annotated using numbers and arrowheads in the gel images) were extracted, trypsinized and identified using a Synapt G2 mass spectrometer. See Table 1 for a listing of the proteins identified by mass spectrometry. C1 to C3 and F1 to F3 are individual specimens from control and felodipine treated lenses, respectively.
Table 1.
The Felodipine treated mouse lenses exhibit decreased levels of certain selected proteins identified by Mass spectrometry.
Figure 5.
Inhibition of LTCCs by felodipine in ex-vivo organ cultured mouse lenses leads to increased Aquaporin-0 Ser235 phosphorylation.
To determine the influence of LTCC inhibition on aquaporin-0 expression and phosphorylation, control lenses and lenses treated with felodipine (10 µM) for 6 (A) and 24 (B) hrs were examined for changes in the levels of total aquaporin-0 (MIP-26), Ser235-phosphorylated aquaporin-0 (Ser235 Phospho-MIP26) and serine phosphorylated proteins in membrane-enriched lens insoluble fractions, This analysis was performed by immunoblotting using the respective antibodies (see Methods) and subsequent quantification with Image J based densitometric analysis (Panels C, D and E). The levels of both Ser235 phosphorylated MIP-26 (aquaporin-0) and serine phosphorylated proteins (∼28 kDa) exhibited significant increases (* P<0.01; n = 4) in felodipine treated (24 hrs) specimens compared to control lenses. The levels of total MIP-26 were found to be unaltered by felodipine treatment. Total MIP-26 was used as a loading control for the phospho-MIP26 analysis. Samples derived from three independent lenses were depicted from both the felodipine and DMSO treated groups. Panel F depicts the distribution of MIP-26 (aquaporin-0) in equatorial sections from felodipine and DMSO treated lenses based on immunofluorescence analysis using a MIP-26 polyclonal antibody. Inserts in Panel F show fiber cell swelling in the felodipine treated specimens (indicated with arrows). Bar indicates magnification. Epi: Epithelium, CF: cortical fibers, NF: Nuclear fibers.
Figure 6.
Increased levels of connexin-50 in LTCC inhibited ex-vivo organ cultured mouse lenses.
To evaluate whether inhibition of LTCCs influences connexin-based gap junction activity, membrane enriched fractions from felodipine and DMSO treated control mouse lenses were examined for changes in connexin-50 protein levels by immunoblot analysis. The levels of connexin-50 were increased significantly in the felodipine treated lenses following 6 hrs (A) and 24 hrs (B) of drug treatment compared to control lenses. Total MIP-26 was used as a loading control for the connexin-50 immunoblots. Lanes 1 to 3 in panel A represent individual specimens from both groups. Panel C shows quantitative changes in connexin-50 in felodipine treated and control specimens, based on densitometric analysis of an n = 4 independent samples. Panel D shows disorganization of connexin-50 distribution in the felodipine treated lens cortical fibers (equatorial sections) relative to control specimens in which connexin-50 distribution is well organized and presents a clustered localization at the fiber cell long arm (see arrows & insert), based on immunofluorescence analysis using connexin-50 polyclonal antibody. Bar indicates magnification. Epi: Epithelium, CF: Cortical fibers, NF: Nuclear fibers.
Figure 7.
Upregulation of Connexin-50 and -46 gene expression in the felodipine treated mouse lenses.
q-RT-PCR was performed to determine whether there is any difference in the transcriptional regulation of connexin gene expression in felodipine treated mouse lenses. For this, total RNA extracted from the felodipine treated (6 and 24 hrs) and control (DMSO treated) lenses was reverse-transcribed and used for q-PCR analyses. Real-time quantification of expression of connexin-50 and -46 genes was normalized to the cycle value (Cycles) of GAPDH. Left panels: plots of log fluorescence units versus cycle number. Right panels: relative percent fold change in connexin-50 and -46 gene expression with felodipine treatment in ex-vivo mouse lens. Fold changes were calculated based on the mean values from triplicate analyses of individual samples. F6 and F24 represent the 6 and 24 hr felodipine treated specimens, respectively.
Figure 8.
Decreased levels of 14-3-3ε, a phospho-serine/threonine protein binding protein in LTCC inhibited mouse lenses.
Analysis of mouse lenses treated with felodipine (10 µM) for 6 hrs (A) and 24 (B) hrs revealed a significant decrease in the levels of 14-3-3ε compared to DMSO treated control lenses, as assessed by immunoblot analysis of lens soluble protein fractions in conjunction with densitometric analysis (Panel C; n = 4). The levels of βB1-crystallin exhibited modest but significant increases in the same samples after a 6 hr treatment with felodipine (A). After 24 hrs of drug treatment (B), however, a significant decrease was noted in the levels of βB1-crystallin in felodipine treated lenses relative to control lenses, based on immunoblot (A & B) and densitometric analyses (D; n = 4). GAPDH staining was used as a loading control for both the analyses.
Figure 9.
Inhibition of LTCCs in ex-vivo organ cultured mouse lenses by felodipine impairs myosin light chain phosphorylation.
To determine the effects of inhibiting LTCC activity by felodipine (10 µM) on MLC phosphorylation and total MLC levels in the lens, felodipine and DMSO (control) treated mouse lenses were analyzed for changes in the levels of phospho-MLC and total MLC by immunoblot analysis. The levels of phospho-MLC exhibited marked and significant decreases in felodipine treated lenses after 6 hrs (A) and 24 hrs (B) of drug exposure, relative to control lenses. Interestingly, the same samples also exhibited significant increases in the level of total MLC following either a 6 hr or a 24 hr exposure to felodipine, as compared to the corresponding control samples (A & B). Panels C and D show quantitative changes in the levels of phospho-MLC and total MLC respectively in felodipine treated lenses based on densitometric analysis of 4 independent specimens. Lanes 1 to 3 represent three independent samples from both control and drug treated groups.
Figure 10.
Decreased intracellular calcium in felodipine treated mouse lens primary epithelial cells.
To confirm the effects of felodipine on intracellular calcium levels, lens epithelial cells derived from P26 mouse lenses were cultured in glass bottom plastic culture chambers and treated with felodipine (10 µM) as described in the Methods section, for 6 or 24 hrs. The drug treated and control cells loaded with FURA-2/AM (4 µM) for 30 min and were rinsed with Hank’s balanced salt solution prior to acquisition of baseline FURA-2AM fluorescence measurements in several individual cells using digital imaging fluorescence microscope at excitation 340/380 nm for 2 min. Following this, while the recoding was ongoing, thapsigargin (1 µM) was added to the cells and FURA-2/AM fluroscence recording continued for an additional 3 min to detect the thapsigargin-mediated depletion of intracellular calcium stores. Based on these analyses and as shown in the figure, only control cells revealed a significant increase in calcium-dependent FURA-2/AM signal from their respective baseline signal. On the other hand cells treated with felodipine for either 6 or and 24 hr did not exhibit any significant increase in calcium signal from their respective baseline values, confirming a marked decrease in total intracellular calcium in the felodipine treated lens epithelial cells compared to the controls. Values represent mean ± Standard deviation based on several independent cell recordings.
Figure 11.
LTCC inhibition by felodipine decreases actin stress fibers and MCL phosphorylation in mouse lens epithelial cells.
Mouse lens primary epithelial cells cultured on gelatin-coated glass cover slips and treated for 6 or 24 hrs with felodipine (10 µM) in the absence of serum revealed a dramatic decrease in actin stress fibers (red) stained with rhodamine phalloidin and phospho-MLC (green) stained with phospho-MLC antibody in conjunction with FITC-conjugated secondary antibody compared to the DMSO treated control cells. Blue stain represents cell nuclei labeled with Hoechst stain. The images are representative of triplicate analyses. Scale bar indicates magnification.
Table 2.
The list of Oligonucleotide Primer sets used in the RT-PCR and q-RT-PCR reactions.