Fig 2: Representative immunoblot of sperm (S1 and S2) and tissue (positive controls; testicle [T], epididymis [E], ovary [Ov] and oviduct [Od]) samples for SLO1 channels (A), and its corresponding peptide competition assay (B). TGX™ (Tris-glycine extended) Stain Free (total protein) and a-tubulin (anti-a-tubulin) were performed as loading controls.

Fig 3: Administration of iberiotoxin followed by 4-aminopyridine shows the same pattern that voltage-gated delayed K+ channels (KV) are the major contributors to the enhanced theophylline-induced K+ currents after testosterone (TES) treatment. (A) Application of theophylline (Theo) 320 µM to single myocytes under control conditions (not incubated with TES) markedly increased the IK+ from -50 mV ahead. This increase was slightly reduced by iberiotoxin (IBTX, 100 nM), a specific blocker of BKCa channels. Successive administration of 4-aminopirydine (4-AP, 3 mM), a blocker of KV channels, to the same cells abolished the remaining Theo-induced increase of IK+ from -40 mV ahead (n = 6). BKCa channels account for 35.17% of the theophylline response, according to an estimation of the area under the curve (AUC) (light gray region), while KV channels account for 64.83%. (dark gray area). (B) When myocytes were incubated with TES 40 nM, the addition of IBTX (100 nM) reduced the Theo-induced K+ current by 31.8% (light gray area), whereas the addition of 4-AP (3 mM) eliminated the theophylline-triggered rise in IK+ enhanced by TES, indicating a major role of KV channels (68.2%, dark gray area) in the androgen action (n = 6). These results also suggest that TES upregulates these proteins. Symbols depict the mean ± S.E.M. In panel (A), ** p < 0.01 when comparing Control (?) vs. Theo (o) groups. †† p < 0.01, comparing Control (?) vs. Theo + IBTX (?). ¶ p < 0.05 comparing Control (?) vs. Theo + IBTX + 4-AP (?). In panel (B), ** p < 0.01 and * p < 0.05 when comparing TES (?) vs. TES + Theo (o) groups. † p < 0.05, †† p < 0.01, comparing TES (?) vs. TES + Theo + IBTX (?). •• p < 0.01, • p < 0.05, comparing TES + Theo (o) vs. TES + Theo + IBTX (?) groups. ¶ p < 0.05, ¶¶ p < 0.01, comparing TES (?) vs. TES + Theo + IBTX + 4-AP (?). Repeated measure analyses of variance were performed, followed by Student–Newman–Keuls’ multiple comparison tests.

Fig 4: Voltage-dependent delayed rectifier K+ (KV) channels are involved in the theophylline-induced IK+ enhancement after testosterone (TES) chronic incubation in guinea pig tracheal smooth muscle cells. (A) Perfusion of theophylline (Theo) 320 µM to single myocytes resulted in a significant increase in IK+ from -40 mV ahead, which was partially reduced by 4-aminopyridine (4-AP, 3 mM), a blocker of KV channels. Subsequent administration of iberiotoxin (IBTX,100 nM), a specific blocker of BKCa channels to the same cells, abolished the remaining theophylline-induced increase in IK+ from 20 mV ahead (n = 6). Calculation of the area under the curve (AUC) shows that KV channels are responsible for 72.17% of the theophylline response (light gray area), while BKCa channels account for 27.83% (dark gray area). (B) The enhancement of theophylline-evoked increase of IK+ by TES from -50 mV ahead, was almost blocked by 4-AP, suggesting a major role of KV channels in the androgen’s action, and the addition of IBTX nearly abolished the IK+ (n = 6). AUC analysis revealed that the contribution of KV and BKCa channels to the TES-induced improved theophylline response was 82.37% (light gray area) and 17.62%, (dark gray area), respectively. These results reveal that the main K+ channels involved in increasing the IK+ triggered by theophylline are the KV channels and suggest that TES upregulates these proteins. Symbols depict the mean ± S.E.M. In panel (A), ** p < 0.01 when comparing Theo (o) vs. Control (?). † p < 0.05, †† p < 0.01, comparing Theo + 4-AP group (?) vs. Control group (?). ¶ p < 0.05, ¶¶ p < 0.01, comparing Theo + 4-AP + IBTX (?) vs. Control (?). In panel (B), ** p < 0.01 when comparing TES + Theo (o) vs. TES (?). † p < 0.05, †† p < 0.01, comparing TES + Theo + 4-AP (?) vs. TES (?). •• p < 0.01, comparing TES + Theo (o) vs. TES + Theo + 4-AP (?). ¶ p < 0.05, ¶¶ p < 0.01, comparing TES + Theo + 4-AP + IBTX (?) vs. TES (?). Repeated measure analyses of variance were performed, followed by Student–Newman–Keuls’ multiple comparison tests.

Fig 5: Testosterone fails to increase KCa1.1 levels in guinea pig airway smooth muscle. In the first column, KCa1.1 immunofluorescence (green color) is shown in guinea pig tracheal tissues without (A) and with (B) 40 nM TES incubation for 48 h. Note that in contrast to KV1.2 and KV1.5, no increase in fluorescence was observed when tissues were exposed to TES. (C) The absence of the primary antibody for KCa1.1 (BKCa channel) showed no fluorescence (NC = negative control). The second column illustrates smooth muscle a-actin (red), and the third column depicts nuclei stained with DAPI (blue). Merged images of the first three panels are shown in the fourth column. The yellow color indicates the colocalization of KCa1.1 and a-actin. (D) Bar graph summarizes the quantification of KCa1.1 levels expressed as mean fluorescence, n = 5. S.E.M. is indicated by error bars. SM = smooth muscle, and EPI = epithelium.