Pregnenolone sulphate- and cholesterol-regulated TRPM3 channels coupled to vascular smooth muscle secretion and contraction

Abstract

Rationale: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in beta cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system.

Objective: To determine the relevance and regulation of TRPM3 in vascular biology.

Methods and results: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. beta-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity.

Conclusions: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

Figure 1

Native TRPM3 expression in vascular smooth muscle cells (VSMCs). (A) RT-PCR analysis of mRNA in fresh human saphenous vein medial layer without (−) and with (+) prior reverse transcription (RT). DNA markers (M) are on the left. The arrow indicates the predicted size of the TRPM3 product (268 bp). (B, C) Cross-sections of intact saphenous vein without (B) and with (C) induction of neointima. (B) I, pre-existing intima; M, medial layer; a, adventitia containing vasa vasorum. (C) NI, neointima; L, lumen. Controls were primary antibody omitted (B) and antibody preadsorbed to antigenic peptide (C). (D) Tissue section containing in vivo injured mouse femoral artery (f) and an uninjured artery (arrow), with the bracket indicating the region of remodeling VSMCs; control staining of the adjacent tissue section was with antibody preadsorbed to antigenic peptide. (B-D) Labeling with anti-TRPM3 antibody is shown in brown colour (B, TM3N1 Ab; C & D, TM3E3 Ab10); scale bars are 100 μm. (E, F) Data from human saphenous vein VSMCs in culture, representative of n=4 (E) and 3 (F). (E) RT-PCR analysis using two PCR primer sets, showing mRNA of TRPM3₁₃₂₅ (i) without TRPM3f insert (ii) (see Supplementary Table I). (F) Immunofluorescence labeling with TM3E3 (green) after transfection with scrambled (control) or TRPM3 siRNA. Cell nuclei were stained with DAPI (blue). The scale bars are 20 μm. The bar chart is mean data for the type of experiments illustrated by the images (n/N = 4/81 for control, 4/100 for TRPM3 siRNA).

Figure 2

Functional activity of endogenous TRPM3 in proliferating VSMCs. Data are based on intracellular Ca²⁺ measurement (A-D) or whole-cell electrophysiology (E, F) applied to human saphenous vein VSMCs. (A) Responses to 25 μmole/L PregS in the presence (+Ca²⁺) or absence (0 Ca²⁺) of 1.5 mmole/L extracellular Ca²⁺ (representative of n/N = 5/30 for each). (B) Example responses to 25 μmole/L PregS after pretreatment with TM3E3 antiserum or its preimmune control. (C) Mean data summarizing effects on responses to 25 μmole/L PregS, 20 μmole/L sphingosine (SPH), 100 μmole/L ATP or 1 μmole/L S1P of pretreatments with TM3E3, anti-TRPC1 antiserum (T1E3) or their preimmune controls (n/N = 7/76 for TM3E3 and 6/47 for T1E3 tests and controls), or transfection with TRPM3 siRNA compared with scrambled siRNA (n/N = 3/18 for each test and control). Each test data set was normalized to its own control. (D) Effect of returning 1.5 mM Ca²⁺ to the extracellular solution following pre-incubation with TM3E3 or its pre-immune serum (N = 6 for each; representative of n=5). Data without Ca²⁺ add-back (0 Ca) are shown for comparison. (E) Example spontaneous current at the indicated voltages, showing effects of extracellular TM3E3 and then 75 μmole/L 2-aminoethoxydiphenylborate (2-APB). (F) Mean data for the type of experiment shown in (E) normalized to currents in paired controls (TM3E3 preadsorbed to its antigenic peptide (+pep.)).

Figure 3

Relationship to secretion from proliferating VSMCs. (A) Absolute interleukin-6 (IL-6) concentration in extracellular medium exposed to TM3E3 (n=9) or TM3E3 preadsorbed to its antigenic peptide (n=7). (B) Effects 10 μmole/L PregS on secretion of IL-6, MMP-9 and hyaluronan (n=8 patients for each condition). (C) Effects 1 μmole/L BAPTA-AM (BAPT.) on IL-6 secretion and its inhibition by 10 μmole/L PregS (n=7 patients for each condition). All data are from human saphenous vein VSMCs.

Figure 4

Relationship to contraction in mouse aorta. (A) PCR analysis of aorta mRNA using primers for TRPM3 (expected product size, 295 bp). PCR products are shown with (+) or without (−) reverse transcription (RT) and DNA markers (M) are on the left. (B-E) Isometric tension recordings. (B, C) Example paired recording showing responses to 10 nmole/L phenylephrine (PE) and 200 μmole/L PregS. Arrows mark PregS wash-out artefacts. Vessel segments were pre-incubated with dialysed and boiled TM3E3 (B, control) or dialysed TM3E3 (C, test ). The verticle scale bars are 0.5 and 0.2 mN (B, C) and the horizontal bars are 5 min. (D) Mean data for the type of experiment illustrated in (B, C) (n=9 pairs). (E) As for (B, C) but excluding TM3E3 and in the continuous presence of 100 nmole/L nicardipine (representative of n=4). Verticle scale bar, 0.5 mN; horizontal scale bar, 10 min.

Figure 5

Sensitivity to neurosteroids. (A, B) Concentration-dependence of responses to PregS (A) or dihydroepiandrosterone sulphate (DHEAS, B) in HEK 293 cells induced to express TRPM3 (Tet+: n/N=3/12, A; n/N=3/9, B) or VSMCs (n/N=4/12, A; n/N=4/16, B). Shaded areas indicate the physiological plasma concentrations.

Figure 6

Chemical screen and identification of cholesterol as a TRPM3 inhibitor. Shown are data from intracellular Ca²⁺ responses in HEK 293 cells without (Tet− in A and C) or with induction of TRPM3 expression (Tet+ in A, and all data in B, D and E). (A) Summary of the outcomes of the chemical screen. Response amplitudes were graded according to the indicated colour scale. Squares A1-2 were for PregS and S13-14 for β-cyclodextrin (βCD). For full decoding of the plate see Supplementary File I. (B) Effect of returning 1.5 mmole/L Ca²⁺ to the extracellular solution following pre-incubation with and without 0.5 mmole/L cholesterol in the presence of 2.5 mmole/L mβCD (N = 4 for each condition). (C) As for (B) but mean data with and without addition of 1.5 mmole/L Ca²⁺, with and without addition of cholesterol to the mβCD, and with (Tet+) and without (Tet−) induction of TRPM3 expression (n/N = 3/12 for each). (D) Example comparison of responses to 25 μmole/L PregS with (+mβCD) or without pre-incubation with 2.78 mmole/L mβCD (control) or with preincubation with 1 mmole/L cholesterol (chol.) delivered on 5 mmole/L mβCD as the carrier. (E) Example responses to 25 μmole/L PregS with (+αCD) and without pre-incubation with 2.78 mmole/L α-cyclodextrin (αCD). (D, E) Each is representative of 3 independent experiments (N = 6 for each condition).

Figure 7

Effects of cholesterol loading on responses in VSMCs. (A, C-E) Data from intracellular Ca²⁺ measurements in 96-well (A, C) or microscope recording systems (D, E). (A) Mean responses to 25 μmole/L PregS with (mβCD) or without (control) pre-incubation with 2.78 mmole/L mβCD or with pre-incubation with 1 mmole/L cholesterol (chol./mβCD) delivered on 5 mmole/L mβCD (n/N=3/18). (B) Bright-field images of Oil Red-O stained cells without and with cholesterol loading for 48 hr. Scale bar, 50 μm. (C) Mean responses to 25 μmole/L PregS or 1 μmole/L ionomycin in cells without (−) and with (+) prior cholesterol loading for 48 hr (n/N = 4/32 for PregS and 4/20 for ionomycin). (D) Example single cell effects of 25 μmole/L PregS without and with prior cholesterol loading for 48 hr (traces are shown for 3 cells: i, ii, iii). (E) Mean data for the type of experiment illustrated in (D) (n/N = 4/28 for control and 4/23 for +chol.). All data are from human saphenous vein VSMCs.