SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels

Figures

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  Figure 1.. Expression of SMIT1 (sodium:myo-inositol transporter 1) in vascular smooth muscle cells (VSMCs). A, Representative immunofluorescence showing the expression of SMIT1 protein in VSMCs isolated from renal arteries (RA, top) and mesenteric arteries (MA, bottom). The right-most parts show the staining in absence of the SMIT1 antibody (No I Ab). The insets show a brightfield image of the cell. Nuclei (DAPI [4',6-diamidino-2-phenylindole, dihydrochloride] staining, blue) are also shown. Scale bar=10 µm (left-most parts) and 5 µm (other parts). B, Quantitative polymerase chain reaction showing the expression of the transcripts encoding for Kv7.4 (kcnq4), Kv7.5 (kcnq5), SMIT1 (slc5a [solute carrier family 5A] 3) and SMIT2 (slc5a11) in VSMCs isolated from renal arteries. Data are expressed using the 2-ΔCq formula (N=5).

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  Figure 2.. Effects of the modulation of SMIT1 (sodium:myo-inositol transporter 1) levels on arterial contractility. A, left, representative images showing the immunostaining of SMIT1 in renal vascular smooth muscle cells (VSMCs) incubated with a scrambled (CTL-mor) or an anti-SMIT1 morpholino oligonucleotide (SMIT1-mor). The insets show brightfield images of the cells. Scale bar=5 µm. The graph shows the quantification of SMIT1 fluorescence intensities measured as corrected total cell fluorescence (CTCF) and expressed as percentage of control (CTL-mor). Data are expressed as mean±SEM. *P<0.05 vs respective controls; n=28 cells for CTL-mor and 30 cells for SMIT1-mor obtained from 3 different preparations (N=3). Right, isometric tension recordings showing the effects of increasing concentration of methoxamine (MO) in segments of renal arteries after 48 h incubation with CTL-mor (black line) or SMIT1-mor (red line). Data are shown as percentage of the contraction obtained with 30 µmol/L MO. Data are expressed as mean±SEM. ***P<0.001; ****P<0.0001 vs controls; N=13 for CTL-mor and 16 for SMIT1-mor. B, left, representative images showing the immunostaining of SMIT1 in renal VSMCs incubated with…

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  Figure 3.. Effects of SMIT1 (sodium:myo-inositol transporter 1)-silencing on K+ channels activators. A, Representative image showing the protocol used for isometric tension recordings. After 2 stimulations with 10 µmol/L methoxamine (MO), segments of renal arteries were incubated for 10 min with K+ channels activators (all used at a concentration of 3 µmol/L) and then stimulated again with MO. B–E, Quantification of the responses to MO in the presence of ML213 (B, N=9), RL-3 (C, N=6), levcromakalim (D, N=5), or NS11021 (E, N=4) in renal arteries incubated with a scrambled (CTL-mor) or an anti-SMIT1 morpholino oligonucleotide (SMIT1-mor). Data are expressed as mean±SEM. and shown as percentage of the contraction to the first stimulation with MO (see A). The responses to the second incubation of MO before the incubation with the activators (MO) are also shown. **P<0.01.

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  Figure 4.. Effects of enhanced SMIT1 (sodium:myo-inositol transporter 1) expression on Kv7 channels blockers. A, top, representative traces showing the effects on isometric tension recordings of the pan-Kv7 blocker linopirdine (3 µmol/L) in renal arteries after 16 h incubation with vehicle (black line) or raffinose plus myo-inositol (R+M; gray line). Bottom, bar graphs showing the contraction to linopirdine (1 and 3 µmol/L) of renal arteries incubated for 16 h with vehicle (control, black bars) or R+M (white bars) in wire-myography experiments. Data are expressed as mean±SEM and shown as percentage of the initial contraction to 60 mmol/L KCl. *P<0.05, **P<0.01; N=10 per experimental group. B, bar graphs showing the effects of methoxamine (3 µmol/L) in the presence of dimethyl sulfoxide (DMSO; vehicle, black bars), 3 µmol/L linopirdine (Lino, white bars) or 1 µmol/L HMR1556 (gray bars) on isometric tension recordings of renal arteries incubated for 16 h with vehicle (control) or R+M. Data are expressed as mean±SEM and shown as percentage of the contraction to 30 µmol/L methoxamine. ***P<0.001; ns=not significant; N=10 per experimental group.

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  Figure 5.. Effects of enhanced SMIT1 (sodium:myo-inositol transporter 1) expression and silencing of Kv7.4 and Kv7.5 in renal arteries. Isometric tension recordings showing the effects of increasing concentration of methoxamine (MO, A) or KCl (B) in segments of renal arteries transfected with a scrambled morpholino (CTL-mor) or 2 morpholinos targeting Kv7.4 and Kv7.5 (Q4/Q5-mor). 32 h after the transfection, arteries were incubated for 16 h with either vehicle (black and gray lines) or 150 mmol/L raffinose plus 1 mmol/L myo-inositol (raffinose plus myo-inositol [R+M]; red and orange lines). Data are shown as percentage of the contraction obtained with 30 µmol/L MO (A) or 120 mmol/L KCl (B). Data are expressed as mean±SEM. *P<0.05; **P<0.01: ****P<0.0001 vs CTL-mor vehicle; ###P<0.001; ####P<0.0001 vs Q4/Q5-mor R+M; N=6 for vehicle incubated arteries and 8 for Q4/Q5-mor arteries. C, Bar graph showing the effects of the Kv7 activator retigabine (RET) in Kv7.4 and Kv7.5-silenced arteries. At the end of the experiments shown in A and B, arteries were challenged twice with 10 µmol/L MO alone (second MO contraction is shown with black bars) and then with MO in the presence of 10 µmol/L RET (white…

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  Figure 6.. Interaction of SMIT1 (sodium:myo-inositol transporter 1) with vascular Kv7 channels. A, Proximity Ligation Assays (PLAs) showing the interaction of SMIT1 with Kv7.4 and Kv7.5 in vascular smooth muscle cells (VSMCs) isolated from renal and mesenteric arteries. The insets show a brightfield image of the cell. Nuclei (DAPI [4',6-diamidino-2-phenylindole, dihydrochloride] staining, blue) are also shown. Scale bar=5 µm. B, Bar graphs showing the interactions of Kv7.4, Kv7.5, and SMIT1 with Kv2.1 and TRPC1 (transient receptor potential C1 channel) in VSMCs isolated from renal arteries incubated for 16 h with vehicle (control, black bars) or 150 mmol/L raffinose plus 1 mmol/L myo-inositol (raffinose plus myo-inositol [R+M], white bars). Data represent the mean number of PLA signals per midcell xy section, expressed as mean±SEM. n=26–36 cells from 3 to 4 rats (N=3–4) per experimental point in 3–4 sessions. *P<0.05 vs respective controls. C, Bar graphs showing the interaction between Kv7.4-SMIT1, Kv7.5-SMIT1, and Kv7.4–Kv7.5 in renal VSMCs. Arteries were incubated with a scrambled morpholino (CTL-mor, black bars), a morpholino targeting Kv7.4 (Q4-mor, white bars), or a morpholino against…

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  Figure 7.. Effects of SMIT1 (sodium:myo-inositol transporter 1) on Kv7.4/Kv7.5-mediated currents. A, Mean current-voltage relationship in Chinese Hamster Ovary cells transfected with plasmids encoding for Kv7.4 and Kv7.5 plus a GFP (green fluorescent protein)-encoding plasmid (black line) or a plasmid encoding for SMIT1 (gray line). Data are expressed as mean±SEM. n=8 per experimental group. B, representative traces showing XE-991 sensitive currents in vascular smooth muscle cells (VSMCs) isolated from renal arteries incubated for 16 h with vehicle (control, black traces) or 150 mmol/L raffinose plus 1 mmol/L myo-inositol (raffinose plus myo-inositol [R+M], gray traces). C, Mean current-voltage relationship of the XE991-sensitive current in VSMCs isolated from renal arteries incubated for 16 h with vehicle (control, black line) or 150 mmol/L raffinose plus 1 mmol/L myo-inositol (R+M, gray line). Data are expressed as mean±SEM. n=7 cells for control and 8 cells for R+M, obtained from 8 animals in 8 experimental sessions. *P<0.05; **P<0.01; ***P<0.001 vs control.