Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids

Abstract

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.

Figure 1. Norgestimate inhibits TRPC3- and TRPC6-mediated Ca²⁺ influx.

OAG-induced changes of [Ca²⁺]_i in fluo-4-loaded TRPC3 CHO cells (A) and TRPC6 HEK-FITR cells (C) were measured in 96-well plates using a fluorometric imaging plate reader. Pre-incubation of cells with 30 µM norgestimate (NG) significantly reduced TRPC3 and −6 mediated Ca²⁺ entry. Representative fluorescence traces are shown. Concentration-response curves for inhibition of TRPC3 (B) and TRPC6 (D) by norgestimate were derived from the area under the fluorescence curves for each given concentration. The solid lines represent the best fit of the data to the Hill model with slopes of n = 1.67 (TRPC3) and n = 0.97 (TRPC6). Means ± SEM of 3 wells (B) or 4 wells (D) are shown. The chemical structure of norgestimate is illustrated in B.

Figure 2. Potent inhibition of TRPC3- and TRPC6-mediated currents by norgestimate.

Effect of 10 µM norgestimate (NG) on whole-cell currents evoked by AlF₄ ⁻ infusion into TRPC3 (A) and TRPC6 (B) expressing cells. Current-voltage (I–V) relationships (left panels) and time course of currents recorded at −70 mV (right panels) are shown. For measurement of I–V curves voltage ramps from −100 to +80 mV were applied at the time points indicated. Background currents were isolated by blocking of TRPC3 and −6 with 100 µM La³⁺. Comparison of the mean inhibition of TRPC3 and −6 by 10 µM norgestimate (C) demonstrates equipotent suppression of both channels. N denotes the number of tested cells. Concentration-response relationship of the inhibition of TRPC6 by norgestimate (D). Means ± SEM of n≥3 experiments per concentration are shown. The line represents the best fit of the data to the dose-response equation with a Hill slope of 1.19.

Figure 3. Effect of norgestimate on TRPC5-mediated currents.

Whole-cell currents evoked by AlF₄ ⁻ infusion into TRPC5 expressing HEK-FITR cells were measured before and after application of 10 µM norgestimate (NG) (A). Current-voltage (I–V) relationships (left panels) and time course of currents recorded at −70 mV (right panels) are shown. For measurement of I–V curves voltage ramps from −100 to +80 mV were applied at the time points indicated. Background currents were isolated by blocking of TRPC5 with 10 µM 2-aminoethoxydiphenyl borate (2-APB). Concentration-response relationship of the inhibition of TRPC5 by norgestimate (B). Means ± SEM of n≥3 experiments per concentration are shown. The line represents the best fit of the data to the dose-response equation with a Hill slope of 1.43.

Figure 4. Norgestimate blocks AVP-activated non-selective cation currents in A7r5 cells independent of vasopressin receptor function.

Effect of 10 µM norgestimate (NG) on whole-cell currents evoked by 100 nM AVP in A7r5 cells (A). I–V relationships recorded at the indicated times (left panels) and time course of currents recorded at −60 mV (right panels) are shown. The I–V curves were obtained during voltage ramps from −100 to +80 mV. Voltage-gated L-type Ca²⁺ channels were blocked by 5 µM nimodipine during the whole experiment. Time-dependent changes of [Ca²⁺]_i in fura-2-loaded A7r5 cells (B). Cells were pre-incubated with or without (control) 10 µM norgestimate (NG) in calcium-free (1 mM EGTA) standard extracellular solution for 5 min before vasopressin receptor stimulation by application of 100 nM AVP. Data represent means ± SEM from 33 cells (control) and 36 cells (norgestimate).

Figure 5. Endothelium-independent relaxation of pre-contracted rat aortic rings by norgestimate.

Representative wire myograph recording (A) illustrating the effect of norgestimate on L-NAME treated intact aortic rings pre-contracted with phenylephrine. Compounds were applied as indicated in the perfusate. Acetylcholine (Ach) was applied to demonstrate the absence of endothelium-dependent vasorelaxation. Higher norgestimate concentrations could not be tested due to the limited solubility of the compound. Concentration-response curve of norgestimate-induced vasorelaxation (B). Norgestimate-induced relaxation was expressed as percentage of the phenylephrine-induced tension prior to norgestimate application. The solid line represents the best fit of the data to the Hill model with: y₀ = −4.26%, y₁ = 84.8%, and n = 0.93. Data represent means ± SEM (n = 6).

Figure 6. Progesterone inhibits TRPC-mediated Ca²⁺ influx.

Time-dependent changes of [Ca²⁺]_i in fluo-4-loaded TRPC3 CHO cells (A), TRPC4- (C), TRPC5- (E) and TRPC6 HEK-FITR (G) cells were measured using a fluorometric imaging plate reader. Representative traces illustrate fluorescence changes induced by application of 200 nM trypsin or 30 µM OAG with or without pre-incubation with 30 µM progesterone (PG). TRPC-independent Ca²⁺ release in C and E was determined in non-induced TRPC4 and −5 HEK-FITR cells. Concentration response curves for inhibition of TRPC3 (B), TRPC4 (D), TRPC5 (F) and TRPC6 (H) by progesterone were derived from the area under the fluorescence curves for each given concentration. The solid lines represent the best fit of the data to the Hill model with slopes of n = 3.6 (TRPC3), 1.15 (TRPC4), 2.5 (TRPC5), and 1.2 (TRPC6). Data represent means of 3 wells (B) or 4 wells (D, F, H). The chemical structure of progesterone is illustrated in B.