Rapid responses to reverse T₃ hormone in immature rat Sertoli cells: calcium uptake and exocytosis mediated by integrin

Abstract

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T₃ (rT₃) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT₃ are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT₃ was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT₃-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT₃ may be mediated by integrin αvβ₃. In addition, it was demonstrated that calcium uptake stimulated by rT₃ is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT₃ also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT₃ modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

Figure 1. Time-course and dose-response curve of rT₃ on Ca²⁺ uptake in Sertoli cells.

(A) Time-course effect of rT₃. Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 30, 60 and 300 s with 0.1 µCi/mL of ⁴⁵Ca²⁺ in the presence or absence of rT₃ (10^-17 M). Means ± S.E.M. n= 4 for all groups. **P < 0.01 and *p < 0.05 compared with control group. (B) Dose-response curve for rT₃ in relation to Ca²⁺ uptake in Sertoli cells. Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 60 s with 0.1 µCi/mL of ⁴⁵Ca²⁺ in the presence or absence of rT₃. Means ± S.E.M. For control and rT₃ (10^-19, 10^-17, 10^-15, 10^-13, 10^-11, 10^-9 and 10^-7 M), n=4 for each group. **P < 0.01 and *p < 0.05 compared with control group.

Figure 2. Influence of RGD peptide on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake in Sertoli cells.

Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 60 s with 0.1 µCi/mL of ⁴⁵Ca²⁺ in the presence or absence of RGD peptide (5 x 10^-7 M) with/without rT₃ (10^-17 M). Means ± S.E.M. For control, rT₃, RGD and rT3 + RGD, n=10 for each group. ***p < 0.001 compared with control group; ###p < 0.001 compared with rT₃ group.

Figure 3. Involvement of ionic channels and intracellular calcium on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake.

(A) Influence of flunarizine, (B) BAPTA-AM and (C) 9-AC on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake in Sertoli cells. Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 60 s with 0.1 µCi/mL of 45Ca2+ in the presence or absence of flunarizine (1 µM), BAPTA-AM (50 µM) and 9-AC (1 µM) with/without rT3 (10-17 M). Means ± S.E.M. For control, n=10; rT3, n=7; flunarizine, n=8; rT3 + flunarizine, n=8; BAPTA-AM, n=8; rT3 + BAPTA-AM, n=6; 9-AC, n=6; rT3 + 9-AC, n=6. ***P < 0.001 and **p < 0.01 compared with control group; ###p < 0.001; ##p < 0.01 and #p < 0.05 compared with rT3 group.

Figure 4. Involvement of kinases proteins on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake in Sertoli cells.

(A) Influence of stearoylcarnitine and (B) PD 98059. Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 60 s with 0.1 µCi/mL of ⁴⁵Ca²⁺ in the presence or absence of stearoylcarnitine (1 µM) and PD 98059 (30 µM) with/without rT₃ (10^-17 M). Means ± S.E.M. For control, n=9; rT₃, n=6; stearoylcarnitine, n=8; rT₃ + stearoylcarnitine, n=9; PD 98059, n=8; rT₃ + PD 98059, n=8. ***p < 0.001 and *p < 0.05 compared with control group; ##p < 0.01 and #p < 0.05 compared with rT₃ group.

Figure 5. Fluorescence images of Sertoli cells stained with quinacrine.

Quinacrine stains individual secretory vesicles in the cell cytoplasm. Sertoli cells in culture were incubated with 3 µM quinacrine for 30 min, washed and photographed under fluorescence illumination immediately (A and C) and at 1 min intervals for 10 min of incubation in the absence or presence of rT₃, respectively (B and D). Incubation of cells with 10^-17 M rT₃ caused fusion of quinacrine-loaded vesicles to the plasma membrane and release of the fluorescent content into the surrounding medium, as seen by the loss of fluorescence from most vesicles located at the cell periphery. This effect was observed after 4 min incubation with rT₃. Also, Sertoli cells were incubated for 10 min with 500 nM of RGD peptide or 1 µM of flunarizine prior to incubation with quinacrine, washed and incubated with 3 µM quinacrine for 30 min. Quinacrine-loaded Sertoli cell cultures, pre-treated with RGD or flunarizine for 10 min, were incubated in the absence or presence of 10^-17 M rT₃ and photographed under fluorescence illumination immediately (E, G, I and K) and at 1-min intervals for 10 min of incubation in the absence or presence of rT₃ (F, H, J and L). Incubation of cells in the presence of 500 nM RGD peptide or 1 µM flunarizine prevented the fusion of quinacrine-loaded vesicles to the plasma membrane and release of the fluorescent content. (A) Control, 0 min. (B) Control, 4 min. (C) rT₃, 0 min. (D) rT₃, 4 min. (E) RGD, 0 min. (F) RGD, 4 min. (G) rT₃ + RGD, 0 min. (H) rT₃ + RGD, 4 min. (I) Flunarizine, 0 min. (F) Flunarizine, 4 min. (G) rT₃ + Flunarizine, 0 min. (H) rT₃ + Flunarizine, 4 min. Experiments were performed 3 times with similar results. Bar = 10 µm.