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Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone.

Majeed Y, Tumova S, Green BL, Seymour VA, Woods DM, Agarwal AK, Naylor J, Jiang S, Picton HM, Porter KE, O'Regan DJ, Muraki K, Fishwick CW, Beech DJ - Cell Calcium (2011)

Bottom Line: Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small.Corticosteroids lacked effect.Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified.

View Article: PubMed Central - PubMed

Affiliation: Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK.

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Steroid effects on agonist-independent TRPM3 activity. Data are from intracellular Ca2+ measurements. (a), (b) Example traces showing the effects of 10 μM progesterone (a) or 50 μM dihydrotestosterone (b) on the Ca2+ signals elicited by addition of 5 mM Ca2+ in cells over-expressing TRPM3 (Tet+). Cells were incubated in Ca2+-free SBS for 30 min and then Ca2+ was ‘added-back’. Responses observed in control (Tet−) cells are shown for comparison (N = 8 for each condition). (c), (d) Mean responses to 5 mM Ca2+ add-back in the presence of vehicle, 10 μM progesterone (+10 prog) (c) or 50 μM dihydrotestosterone (+50 DhT) (d) (N/n = 24/3 for each).
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fig0030: Steroid effects on agonist-independent TRPM3 activity. Data are from intracellular Ca2+ measurements. (a), (b) Example traces showing the effects of 10 μM progesterone (a) or 50 μM dihydrotestosterone (b) on the Ca2+ signals elicited by addition of 5 mM Ca2+ in cells over-expressing TRPM3 (Tet+). Cells were incubated in Ca2+-free SBS for 30 min and then Ca2+ was ‘added-back’. Responses observed in control (Tet−) cells are shown for comparison (N = 8 for each condition). (c), (d) Mean responses to 5 mM Ca2+ add-back in the presence of vehicle, 10 μM progesterone (+10 prog) (c) or 50 μM dihydrotestosterone (+50 DhT) (d) (N/n = 24/3 for each).

Mentions: Although nifedipine is chemically distinct from pregnenolone sulphate, we could not be sure that it activated TRPM3 independently of the pregnenolone sulphate binding site. Therefore, we also sought TRPM3 activity in the absence of an exogenous agonist. Over-expressed human TRPM3 has been shown to have constitutive activity [9]. However, in our tetracycline-inducible TRPM3 cells (in the presence of 1.5 mM extracellular Ca2+) such activity was not evident as a basal Ca2+ signal (Fig. 1b; Supplementary Fig. IV) or ionic current (Supplementary Fig. IV). Therefore we used a Ca2+ add-back protocol in which 5 mM Ca2+ was added to cells after a period in Ca2+-free solution (Fig. 6). This protocol revealed a Ca2+ response in TRPM3-expressing as well as control cells but the response was significantly larger in the presence of TRPM3 (Supplementary Fig. V). Therefore we used this signal to investigate effects of progesterone and dihydrotestosterone in the absence of an exogenous TRPM3 agonist. Progesterone inhibited the signal in TRPM3-expressing but not control cells (Fig. 6a, c), whereas dihydrotestosterone had no effect in either set of cells (Fig. 6b, d). The data support the hypothesis that progesterone inhibited TRPM3 independently of competition with an exogenous agonist, apparently acting as a mode-independent inhibitor. Dihydrotestosterone, by contrast, had an inhibitory effect that depended on the presence of the agonist pregnenolone sulphate.


Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone.

Majeed Y, Tumova S, Green BL, Seymour VA, Woods DM, Agarwal AK, Naylor J, Jiang S, Picton HM, Porter KE, O'Regan DJ, Muraki K, Fishwick CW, Beech DJ - Cell Calcium (2011)

Steroid effects on agonist-independent TRPM3 activity. Data are from intracellular Ca2+ measurements. (a), (b) Example traces showing the effects of 10 μM progesterone (a) or 50 μM dihydrotestosterone (b) on the Ca2+ signals elicited by addition of 5 mM Ca2+ in cells over-expressing TRPM3 (Tet+). Cells were incubated in Ca2+-free SBS for 30 min and then Ca2+ was ‘added-back’. Responses observed in control (Tet−) cells are shown for comparison (N = 8 for each condition). (c), (d) Mean responses to 5 mM Ca2+ add-back in the presence of vehicle, 10 μM progesterone (+10 prog) (c) or 50 μM dihydrotestosterone (+50 DhT) (d) (N/n = 24/3 for each).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3275754&req=5

fig0030: Steroid effects on agonist-independent TRPM3 activity. Data are from intracellular Ca2+ measurements. (a), (b) Example traces showing the effects of 10 μM progesterone (a) or 50 μM dihydrotestosterone (b) on the Ca2+ signals elicited by addition of 5 mM Ca2+ in cells over-expressing TRPM3 (Tet+). Cells were incubated in Ca2+-free SBS for 30 min and then Ca2+ was ‘added-back’. Responses observed in control (Tet−) cells are shown for comparison (N = 8 for each condition). (c), (d) Mean responses to 5 mM Ca2+ add-back in the presence of vehicle, 10 μM progesterone (+10 prog) (c) or 50 μM dihydrotestosterone (+50 DhT) (d) (N/n = 24/3 for each).
Mentions: Although nifedipine is chemically distinct from pregnenolone sulphate, we could not be sure that it activated TRPM3 independently of the pregnenolone sulphate binding site. Therefore, we also sought TRPM3 activity in the absence of an exogenous agonist. Over-expressed human TRPM3 has been shown to have constitutive activity [9]. However, in our tetracycline-inducible TRPM3 cells (in the presence of 1.5 mM extracellular Ca2+) such activity was not evident as a basal Ca2+ signal (Fig. 1b; Supplementary Fig. IV) or ionic current (Supplementary Fig. IV). Therefore we used a Ca2+ add-back protocol in which 5 mM Ca2+ was added to cells after a period in Ca2+-free solution (Fig. 6). This protocol revealed a Ca2+ response in TRPM3-expressing as well as control cells but the response was significantly larger in the presence of TRPM3 (Supplementary Fig. V). Therefore we used this signal to investigate effects of progesterone and dihydrotestosterone in the absence of an exogenous TRPM3 agonist. Progesterone inhibited the signal in TRPM3-expressing but not control cells (Fig. 6a, c), whereas dihydrotestosterone had no effect in either set of cells (Fig. 6b, d). The data support the hypothesis that progesterone inhibited TRPM3 independently of competition with an exogenous agonist, apparently acting as a mode-independent inhibitor. Dihydrotestosterone, by contrast, had an inhibitory effect that depended on the presence of the agonist pregnenolone sulphate.

Bottom Line: Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small.Corticosteroids lacked effect.Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified.

View Article: PubMed Central - PubMed

Affiliation: Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK.

Show MeSH
Related in: MedlinePlus