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1.
Figure 2

Figure 2. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone inhibits [Ca2+]i oscillations induced by TCR ligation. (A) [Ca2+]i responses from four representative B3Z cells activated by contact with SIINFEKL-presenting K897 cells illustrate that [Ca2+]i oscillations were reversibly inhibited by the application of 50 μM progesterone to the bath (bar). (B) [Ca2+]i oscillations in four B3Z cells activated by settling onto coverslips coated with anti–CD3-ε antibodies in the absence (a) or presence (b) of 50 μM progesterone.

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
2.
Figure 7

Figure 7. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone blocks CTX-resistant KV channels. Pipette solution as in Fig. 5. (A) Current responses of the total KV current (•) and the CTX-resistant current (▪) to repetitive voltage pulses from −80 to +30 mV separated by 1 s. At this rate of pulsing, Kv1.3 channels undergo cumulative inactivation, as shown by normalized peak current amplitudes in the absence of CTX (▪). With 100 nM CTX present (•), the remaining current does not decline during repetitive pulsing. (B) Progesterone or RU 486 (50 μM) blocks the KV component. (C) Progesterone blocks the CTX-resistant component of KV current more than RU 486. 100 nM CTX was preapplied in Ringer in order to block Kv1.3 channels.

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
3.

Figure 3. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone reduces the [Ca2+]i plateau evoked by TG stimulation in human T cells. (A) Average [Ca2+]i is plotted against time (n = 76) before and during stimulation with 1 μM TG (arrow). TG stimulated Ca2+ influx, resulting in a stable rise in [Ca2+]i. Addition of 30 μM progesterone (bar) reversibly reduced the [Ca2+]i plateau obtained after TG stimulation. (B) The concentration dependence of the reduction of [Ca2+]i by progesterone is plotted for TG-stimulated cells. Calcium levels were normalized by subtracting the resting [Ca2+]i and dividing [Ca2+]i in the presence of progesterone and TG by [Ca2+]i in the presence of TG alone. Data are presented as mean ± SD and were fitted to a Hill equation illustrated by the smooth curve (IC50 = 28 ± 2.7 μM and n = 1.3 ± 0.2).

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
4.
Figure 6

Figure 6. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Inactivation enhances KV block by progesterone. (A) KV currents were elicited by steps to +30 mV from holding potential of −80 mV (1, 3) or −50 mV (2, 4) in the absence (1, 2) or presence (3, 4) of 30 μM progesterone. Solutions used are as in Fig. 5. (B) Use-dependent block of voltage-gated K+ currents. Current responses were elicited by repetitive voltage pulses from −80 to +30 mV separated by 20 s. Normalized peak current amplitudes in the absence (▪) or presence (▾) of 67 μM progesterone were plotted against time. (C) Time course of recovery from inactivation in a B3Z cell before and during application of 60 μM progesterone. Pairs of 200-ms pulses to +30 mV were applied from a holding potential of −80 mV. The graph shows the ratio of peak current during the second pulse to the peak current during the first pulse, plotted as a function of the time interval between the pulses. Data were fitted by single exponentials with time constants of 4 s for control (▪) and 39 s for progesterone (▾).

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
5.

Figure 8. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Concentration-dependent inhibition of KCa current in B3Z cells by progesterone. (A) KCa currents were activated by dialyzing the cell with a solution containing (in mM) 140 K+ aspartate, 2 MgCl2, 7.8 CaCl2, 10 EGTA, 5 Hepes (pH 7.2). The nominal free Ca2+ concentration of this solution was 1 μM, assuming a dissociation constant for EGTA and Ca2+ of 10−7 at pH 7.2. Ca2+-activated K+ currents were evoked by voltage ramps of 200-ms duration from −120 to +50 mV every 30 s. Application of progesterone at different concentrations (indicated at the right of each trace) inhibited the KCa current. (B) Concentration–response curve for progesterone block of KCa currents (•). The slope conductance between −100 and −60 mV was used as a measure of the KCa conductance to avoid contamination by KV currents. Data were normalized to the conductance measured in the absence of progesterone and presented as mean ± SD. The line represents the fit to a Hill equation with IC50 = 113 μM and n = 1.2. Block of KCa channels by 60 μM RU 486 (□) is shown for comparison. (C) Comparison of progesterone and RU 486. Slope conductance values at −80 mV illustrate activation and block of the KCa current by RU 486 and progesterone, each applied at 60 μM.

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
6.

Figure 5. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone blocks KV channels. Whole-cell currents were measured in human T cells (A) and B3Z cells (B) during 200-ms voltage pulses from a holding potential of −80 mV to +30 mV applied every 30 s. The pipette solution contained (in mM): 160 K+ aspartate, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 Hepes (pH 7.2). Currents are shown before and during bath application of 10, 30, or 100 μM progesterone. (C) Concentration dependence for the reduction of KV currents by progesterone. Peak current amplitudes in human T cells (▵) and B3Z cells (□) were analyzed and plotted against progesterone concentration. Data points for B3Z and human T cells overlie each other at most concentrations. For human T cells, the current at the end of the 200-ms pulse (▴) was also plotted. To determine the effect of depolarization on progesterone block, Em was held at −50 mV (○). Data were normalized to control currents measured in the absence of progesterone and presented as mean ± SD. The line represents a fit using a Hill equation with an IC50 of 29 ± 2 μM and n = 2.1 ± 0.4.

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
7.
Figure 9

Figure 9. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone does not affect CRAC or Cl channels. (A) Whole-cell recordings of Ca2+ current through CRAC channels measured during voltage ramps from −100 to +40 mV for a duration of 200 ms. The pipette solution contained (in mM) 128 Cs+ aspartate, 12 BAPTA, 0.9 CaCl2, 3.16 MgCl2, Hepes (pH 7.2). The external solution had the following composition (in mM): 150 NaMeSO3, 20 CaCl2, 10 glucose, 10 Hepes (pH 7.4). (B) Time-dependent changes in the amplitude of the current measured at −80 mV. Arrows, Representative currents recorded before store depletion (1), and after maximal induction activation of CRAC channels, while superfusing cells with control solution (2) or with an external solution containing 30 μM progesterone (3). (C) Whole-cell recordings of swelling-activated Cl current (ICl) measured during voltage ramps from −120 to +40 mV for a duration of 200 ms. The pipette solution contained (in mM) 140 Cs+ aspartate, 2 MgCl2, 4 MgATP, 1 CaCl2, 10 EGTA, 10 Hepes (pH 7.2). For the induction of ICl, the pipette solution was made hypertonic (390–400 mosmol) by the addition of 100 mM glucose. Currents are shown before the onset of cell swelling (1), and after maximal induction of ICl, while superfusing cells with control solution (2) or with an external solution containing 50 μM progesterone (3). (D) Time-dependent changes in the slope conductance measured at the reversal potential for Cl.

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
8.
Figure 4

Figure 4. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone reduces the driving force for Ca2+ influx. (A) Current-clamp recordings using the perforated-patch technique were performed in B3Z cells to determine the effects of progesterone on Em. This panel presents a representative single-cell recording. The addition of 1 μM TG (arrow) hyperpolarized the cell from the resting potential (−60 mV) to near the K+ equilibrium potential (∼−80 mV), enhancing the driving force for Ca2+ influx. Subsequent application of 50 μM progesterone depolarized the cell to −26 mV, resulting in a reduction of the driving force for Ca2+ influx. For perforated-patch recordings, the tips of the pipettes were filled with the following solution (in mM): 120 K2SO4, 16 KCl, 5 MgSO4, 10 Hepes (pH 7.2). A stock solution of nystatin in DMSO (25 μg/ml) was prepared daily and subsequently diluted in the pipette solution to a final concentration of 100 μg/ml. After sonication, this solution was used for backfilling the pipettes as described previously (reference 31). (B) Measurements of average [Ca2+]i from B3Z cells (n = 37). After the addition of 1 μM TG (arrow), [Ca2+]i rose from a resting level of 70 ± 30 nM to a plateau of 1.3 ± 0.4 μM. Progesterone (30 μM) reduced the [Ca2+]i to approximately half of plateau concentration, an effect that was completely reversed by the addition of 2 μM valinomycin. Application bars, The additions of progesterone and valinomycin to the bath.

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.
9.

Figure 1. From: A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes .

Progesterone inhibits NF-AT– mediated gene expression in B3Z cells. (A) The concentration-dependent inhibition of lacZ expression in B3Z cells by progesterone (•) or RU 486 (□) was measured in a multiwell fluorescence plate reader using MUG as a substrate for β-galactosidase. The cells were stimulated for 4 h with 1 μM TG plus 50 nM PMA. For each experiment, triplicate samples were corrected for background fluorescence and normalized for control lacZ expression. Data are presented as mean ± SD (n = 10), and were fitted to a Hill equation of the form where y = the fraction of control lacZ expression with a maximum level represented by ymax, [X] = the concentration of progesterone, IC50 = the dissociation constant, and n = the Hill coefficient. The curve represents a Hill equation with an IC50 value of 22 ± 2.1 μM and n = 1.7 ± 0.3. The effects of progesterone were not due to nonspecific toxicity, since after treatment with 30 μM progesterone, >95% of the cells stained with vital stain acetoxy-methoxy calcein and <5% stained with propidium iodide, a dye that is excluded from live cells. (B) Application of 30 μM progesterone reduced lacZ expression when B3Z cells were stimulated for 4 h by 1 μM TG alone, a combination of 1 μM TG plus 50 nM PMA (TG + PMA), immobilized anti–CD3-ε antibodies, or K897 cells presenting SIINFEKL. Fluorescence readouts from the multiwell plate reader in arbitrary units (a.u.) are presented as mean ± SD (white bars, stimulation alone; hatched bars, stimulation plus 30 μM progesterone). *Significance was determined with one-tail t tests (P < 0.0001).

George R. Ehring, et al. J Exp Med. 1998 November 2;188(9):1593-1602.

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