Pertussis Toxin Nullifies the Depolarization of the Membrane Potential and the Stimulation of the Rapid Phase of 45Ca2+ Entry Through L-type Calcium Channels that are Produced by Follicle Stimulating Hormone in 10- to 12-Day-Old Rat Sertoli Cells

The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca²⁺ into cells and to the Ca²⁺-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of ⁴⁵Ca²⁺ uptake and [¹⁴C]-methyl aminoisobutyric acid ([¹⁴C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca²⁺ channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of ⁴⁵Ca²⁺ entry and [¹⁴C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca²⁺ channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I.

Introduction

Follicle stimulating hormone (FSH) regulates a vast array of Sertoli cell functions by acting through its transmembrane G protein-coupled receptor (GPCR). The complexity of signaling pathways activated by FSH after receptor binding has been emphasized in a number of recent studies (Griswold, 1998; Cheng and Mruk, 2002; Silva et al., 2002; Walker and Cheng, 2005; Loss et al., 2007; Ulloa-Aguirre et al., 2007). FSH induces the rapid activation of multiple signaling cascades, especially cAMP-adenylate cyclase-protein kinase A signaling, that impact diverse biological processes in Sertoli cells, such as those involving Ca²⁺ uptake and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) (Marinissen and Gutkind, 2001; Cunnigham et al., 2003; Meroni et al., 2004; Ulloa-Aguirre et al., 2007). Some of these effects occur very rapidly, within seconds to minutes, as illustrated by the effect of FSH on membrane potential (MP; Wassermann et al., 1992b) and Ca²⁺ uptake through L-type voltage-dependent Ca²⁺ channels (VDCC) in the Sertoli cells of immature rat testes (Grasso and Reichert Jr., 1989 Gorczynska and Handelsman, 1991; Gorczynska et al., 1994; Sharma et al., 1994). The FSH stimulatory actions on Ca²⁺ uptake (Dahia and Rao, 2006) and on insulin-like growth factor type I (IGF-I) synthesis (Khan et al., 2002; Meroni et al., 2004) differ between pre-pubertal and pubertal rat testes, being more active during the proliferative phase of the Sertoli cells. It has also been reported that during this phase FSH greatly induces the Ca²⁺-dependent Na⁺-amino acid co-transport (Wassermann et al., 1992a).

In immature rat testes, FSH also induces biphasic MP changes in Sertoli cells that are specific to the rapid effects of the hormone: a very short hyperpolarization phase (seconds) followed by a prolonged depolarization phase (more than 6 min) (Wassermann et al., 1992b). The hyperpolarization is blocked by tolbutamide, an inhibitor of ATP-sensitive K⁺ channels ( ) (Loss et al., 2007). Forskolin [an adenylate cyclase (AC) activator] and isoproterenol (a β-adrenergic agonist) also produce a rapid hyperpolarization of the immature Sertoli cell membrane. These actions are inhibited by the channel-blocking agent, glibenclamide (Jacobus et al., 2005). FSH-induced depolarization is also prevented by verapamil, a blocker of L-type VDCCs. These findings indicate that FSH-induced depolarization is related to the uptake of Ca²⁺ through VDCCs (Gorczynska and Handelsman, 1991; Wassermann et al., 1992b). The rapid effect of FSH on the immature Sertoli cell membrane has been widely reported (Grasso and Reichert Jr., 1989; Gorczynska and Handelsman, 1991; Wassermann et al., 1992b; Sharma et al., 1994; Dahia and Rao, 2006; Loss et al., 2007; Ulloa-Aguirre et al., 2007), but there is as yet no satisfactory explanation for its mechanism of action.

Follicle stimulating hormone stimulation of Sertoli cells leads to an increase of intracellular Ca²⁺ through a biphasic kinetic process (Grasso et al., 1992). The first phase, called the “surge phase”, is very rapid (seconds) and is associated with a Ca²⁺ current through L-type VDCC (Wassermann et al., 1992b). The second phase, known as the “sustained phase” is slower (minutes to hours) and involves mobilization of intracellular Ca²⁺ associated with cAMP production (Grasso and Reichert, 1990; Gorczynska et al., 1994; Sharma et al., 1994).

Ulloa-Aguirre et al. (2007) reported that [Ca²⁺]i increases may occur in either a cAMP-dependent or cAMP-independent manner. During the sustained phase, cAMP appears to increase intracellular calcium by promoting calcium mobilization from intracellular stores, whereas in the cAMP-independent surge phase, different G proteins or βγ heterodimers might be involved.

The mechanism of the FSH-stimulated rapid ⁴⁵Ca²⁺ current/inflow (1 min; Dahia and Rao, 2006) in the surge phase is not clear. However, this phenomenon is an important component of Sertoli cell membrane signaling during the proliferative phase. Kinetic analysis of FSH-stimulated calcium-dependent [¹⁴C]-MeAIB uptake in Sertoli cells revealed that [¹⁴C]MeAIB transport is very rapid (seconds), and is caused by an increase in V_max, indicating a fast calcium-dependent increase in the activity of functional transporters (Silva et al., 2002).

Earlier studies established that Sertoli cells secrete IGF-I following FSH stimulation. IGF-I synthesis only occurs in the Sertoli cells of immature animals. Both FSH and IGF-I are important determinants of testicular development and Sertoli cell function. It is currently thought that FSH affects the proliferation and differentiation of Sertoli cells from 10-day-old rats by mediating IGF-I production, at least in part (Khan et al., 2002). Some of the biological effects of IGF-I in Sertoli cells depend on activation of the PI3K/Akt signaling pathway. FSH not only stimulates the secretion of IGF-I in immature rat Sertoli cells, but it also enhances the effects of IGF-I on the PI3K/Akt pathway (Khan et al., 2002). However, it has also been reported that FSH can activate PKB/Akt independently of IGF-I in a PI3K-dependent manner in Sertoli cells from 20-day-old rats (Meroni et al., 2004).

Hormone receptors are known to bind more than one type of G protein (Hubbard and Hepler, 2006; Nechamen et al., 2007). For example, the β2 adrenergic receptor activates AC via Gs stimulation and can also powerfully stimulate Gi in murine cardiac myocytes (Ford et al., 1998; Xiao et al., 1999). Furthermore, in HEK 293 cells, adrenergic stimulation of MAPK by the β2 adrenergic receptor is mediated by the βγ subunits of pertussis toxin (PTX)-sensitive G protein (Daaka et al., 1997).

Gorczynska et al. (1994) reported that the relative roles of Gs and Gi in FSH signal transduction in Sertoli cells were unclear. They observed that the rapid (60–240 s) Ca²⁺ increase produced by FSH in isolated Sertoli cells from immature rats was significantly inhibited by pre-incubation with 1 mg/l PTX for 90 min (Figure 4 from Gorczynska et al., 1994). They proposed that, in addition to FSH stimulating AC via coupling to a Gs protein, the FSH receptor may also be linked to a Gi protein (Gorczynska and Handelsman, 1991).

Other authors have also reported the presence of active Gi subunits in Sertoli cells (Monaco et al., 1988; Krantic and Benahmed, 2000). Crépieux et al. (2001) demonstrated that FSH activates the ERK/MAPK pathway following coupling of the FSH receptor to both the Gs and Gi heterotrimeric proteins in primary cultures of Sertoli cells from 5- to 12-day-old rats. This dual signaling mechanism could explain the different electrophysiological effects of FSH on Sertoli cells during the proliferative phase. FSH action on Gs proteins could induce hyperpolarization of the MP, probably through the conversion of ATP to cAMP, opening channels (Nakashima and Vanhoutte, 1995), whereas the depolarization phase associated with Ca²⁺ entry (current) through the L-type VDCC (Wassermann et al., 1992b) and the consequent increase on the MeAIB transport (Wassermann et al., 1992a) could be caused by Gi protein activation.

In light of these previous studies, we investigated the action of PTX on (a) FSH-induced changes in the MP of Sertoli cells from 10- to 12-day-old rats; (b) the rapid initial phase (seconds) of Ca²⁺ inflow or entry trough L-type VDCCs stimulated by FSH, and (c) [¹⁴C]-MeAIB transport by the Ca²⁺-dependent A system that is activated by FSH. The possible mediation of IGF-I in these phenomena was also investigated.

Materials and Methods

Follicle stimulating hormone, IGF-I, wortmannin, PTX, verapamil, tolbutamide, and forskolin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). JB1 was purchased from Bachem Bioscience, Inc. (King of Prussia, PA, USA). ⁴⁵Ca²⁺ (specific activity 444 GBq/g), and α-[1-¹⁴C] methylamino isobutyric acid ([¹⁴C]-MeAIB, specific activity 1.85 GBq/mmol) were acquired from Perkin ElmerNEN^® (Waltham, MA, USA). The anti-IGF-I polyclonal antibody we used was acquired from the National Hormone and Peptide Program (NHPP) of the National Institute of Diabetes and Digestive and Kidney Disease (NIDDKD-NIH) at the UCLA Medical Center (Torrance, CA, USA).

The experimental animals were immature, 10- to 12-day-old Wistar rats. The animals were bred in our animal facility and housed in an air-conditioned room (approximately 24°C) with controlled lighting (lights on from 06:00 to 20:00). Pelleted food (Purina, Nutripal, Porto Alegre, Rio Grande do Sul, Brazil) and tap water were available to the mothers ad libitum. The suckling rats were kept with their mothers until they were sacrificed by cervical dislocation.

Solutions and Drugs Used

Krebs Ringer bicarbonate buffer solution (KRb): NaCl (146 mM), KCl (4.7 mM), KH₂PO₄ (1.2 mM), NaHCO₃ (25 mM), MgSO₄·7H₂O (1.2 mM), CaCl₂·2H₂O (2.5 mM), Glucose (5.5 mM). This solution was gassed with O₂:CO₂ (95:5, v/v) until it reached pH 7.4. Stock solutions of wortmannin and tolbutamide were prepared in dimethyl sulfoxide (DMSO) and stored at −20°C until required. These stock solutions were diluted in KRb to achieve the final concentration at the time of use. The final concentration of DMSO did not exceed 0.1% or affect the analyzed parameters. The drugs concentrations used in each experiment are included in the figures and legends.

Electrophysiological Experiments

In each experiment, whole testes were decapsulated and carefully stretched with two callipers, exposing 3–10 undisrupted seminiferous tubules. They were then fixed to the bottom of a superfusion chamber, incubated with 1 ml/min KRb buffer with glucose (5 mmol/l), pH 7.4, at 34°C and equilibrated with O₂:CO₂ (95:5; v/v). Standard single microelectrode recording was performed according to the method described in von Ledebur et al. (2002). Microelectrode borosilicate pipettes were filled with KCl (3 M) and had a tip resistance of 15–25 MΩ. Intracellular recording was amplified using an intra 767 WPI (World Precision Instruments, Inc., USA). Square current pulses of 0.5 nA, 0.5 Hz, and 250-ms duration were applied through the intracellular electrode to estimate membrane resistance (R₀) using the S48 stimulator (Grass Instrument, West Warwick, RI, USA). To register the recording, we used an oscilloscope (Tektronix, 2 Channel Digital Oscilloscope TDS 210, Beaverton, OR, USA) and Wavestar Lite Version 1.0.10 software. FSH (4 mU/ml), IGF-I (100 or 25 ng/ml) and forskolin (100 nM) were applied topically to the bath after the resting potential had stabilized for at least 2 min. Tolbutamide (10 μM) or verapamil (100 μM) solutions were perfused for 10 min before the topical application of FSH or IGF-I. After decapsulation, testes were pre-incubated in KRb buffer with glucose (5 mM/l) and 1 μg/ml of PTX for 60 min, processed as described above and fixed in the superfusion chamber before the MP was recorded and the FSH was applied topically. Each treatment was repeated at least five times with different cells, and variations in MP and input membrane resistance (R₀) were recorded. The results are given as mean ± SEM.

⁴⁵Ca²⁺ uptake experiments

Testes were removed and one gonad from each rat, alternately the left or right, was used as the experimental gonad; the contralateral gonad was used as the control. The testes (n = 5 in each group) were weighed, decapsulated and pre-incubated in KRb with ⁴⁵Ca²⁺(2.7 kBq/sample) for 60 min in a Dubnoff metabolic incubator to equilibrate intra- and extracellular ⁴⁵Ca²⁺ levels until they reached a plateau, which was carried out at 34°C, pH 7.4, with O₂:CO₂ (95:5; v/v). Following equilibration, the gonads were incubated for 2 min in KRb with ⁴⁵Ca²⁺, with or without FSH (4 mU/ml), IGF-I (25 ng/ml), or forskolin (100 nM). In some experiments, testes were pre-incubated for 10 min with JB1 or verapamil, or for 1 h with wortmannin or PTX. To end the experiment and stop calcium flux, 1 ml of cold buffer (0°C) containing lanthanum chloride (LaCl₃) (10 mM) was added to the samples (Batra and Sjogren, 1983). This solution was calcium-free and, in addition to LaCl₃, contained (in mM): NaCl, 127.5; KCl, 4.6; MgCl₂, 1.2; LaCl₃, 10; HEPES, 10; and glucose, 11 (Batra and Sjogren, 1983). The supernatant was preserved, and the testes were removed to screw-cap tubes containing 1 ml of distilled water and stored at −20°C for further analysis. The testes were frozen and subsequently boiled. Aliquots of 100 μl were taken from each sample and placed in Aquasol 2^® (Perkin Elmer, Inc, USA) for measurement of radioactivity using an LKB rack beta liquid scintillation spectrometer, model 1215 (LKB-Producer AB, Bromma, Sweden). The counting efficiency was 85–90%.

[¹⁴C]-MeAIB Transport Experiments

Testes were processed as described above. The samples (n = 5 in each group) were weighed, decapsulated and pre-incubated in KRb buffer for 60 min in a Dubnoff metabolic incubator at 34°C, pH 7.4, and gassed with O₂:CO₂ (95:5; v/v). The gonads were then incubated for 45 min in KRb with [¹⁴C]-MeAIB (2.7 kBq/sample), with or without FSH (4 mU/ml) or IGF-I (different doses, see Figure 6B). In experiments utilizing wortmannin, PTX, or the anti-IGF-I antibody, the testes were pre-incubated for 60 min and then incubated in the presence of the appropriate drug. The supernatant was preserved, and the testes were removed to screw-cap tubes containing 1 ml of distilled water and stored at −20°C until further analysis as above.

Statistical analyses were performed using unpaired Student’s t-test, a two-way ANOVA with Bonferroni’s post test or a two-way ANOVA of repeated measures with Bonferroni’s post test, which were carried out using GraphPad InStat version 3.01, 32 bit for Windows 95/NT (GraphPad Software, San Diego, California, USA, www.graphpad.com). Differences were considered to be significant when p < 0.05. In brackets, number of samples from one or more experiments.

Results

Basal Electrophysiological Values of 10- to 12-Day-Old-Rats

In our experimental conditions, the basal electrical characteristics of impaled Sertoli cells were as follows: resting potential −44 ± 1.1 mV (n = 73) and membrane resistance 10 ± 0.5 MΩ (n = 73). These conditions were stable for at least 30 min before the experiment (Figure 1A).

FIGURE 1

Figure 1. Effect of 1 h of PTX pre-incubation (1 μg/ml) on the action of FSH (4 mU/ml) on the membrane potential of 10- to 12-day-old rat Sertoli cells. Register of a Sertoli cell with resting potential of E_m = −44 mV (A). Typical response of cells treated with FSH without (B) and with PTX (C). (D) Changes of membrane potential produced by FSH application with or without PTX. Statistical analysis: two-way ANOVA followed by Bonferroni post test, **p < 0.001; n = 5 in each treatment (FSH or FSH plus PTX) from five separated experiments.

Effect of PTX on the Electrophysiological Action of FSH on Sertoli Cells

Figure 1B shows the response to a topical application of 4 mU/ml of FSH on the seminiferous tubules in the superfusion chamber (without stopping the flow) after at least 2 min of MP stabilization. FSH produces a biphasic effect characterized by rapid hyperpolarization followed by depolarization. Pre-incubation (60 min) with PTX prevents the depolarization phase induced by FSH (Figure 1C). Figure 1D shows the time course of the action of FSH and FSH plus PTX on the MP of Sertoli cells. The statistical differences in all times of the depolarization response were highly significant (**p < 0.001; n = 5). This depolarization was also previously shown to be inhibited by verapamil, an L-type VDCC blocker (Wassermann et al., 1992b; Loss et al., 2007).

FSH and Forskolin Induced Hyperpolarization in Sertoli Cell Membranes Related to Channels

Activation of the AC-cAMP pathway by forskolin produces hyperpolarization of Sertoli cell membranes (p < 0.001; n = 5) (Figures 2C,D). The hyperpolarization produced by forskolin was inhibited by glibenclamide, which blocks channels, as was previously reported by Jacobus et al. (2005). FSH-induced rapid hyperpolarization phase was also inhibited by pre-treatment with tolbutamide, an inhibitor of channels (**p < 0.001; n = 5) (Figures 2A,B).

FIGURE 2

Figure 2. channel involvement in the hyperpolarization produced by FSH and forskolin on the Sertoli cell membrane. Pre-treatment with tolbutamide inhibited the rapid hyperpolarization response without affecting the depolarization phase, register of a representative cell (A). Changes produced by tolbutamide on the FSH response (B). Topical application of forskolin (100 nM) produced hyperpolarization on Sertoli cell membrane (C). Comparison of the FSH and forskolin action on Sertoli cell membrane potential (D). **p < 0.001; n = 5 in each treatment (FSH or FSH plus tolbutamide) from five separated experiments.

Electrophysiological Effect of IGF-I on the Sertoli Cell Membrane

The topical administration of IGF-I (25 ng/ml) on seminiferous tubules from immature rat testes induced an immediate depolarization in the MP of impaled Sertoli cells (*p < 0.05; n = 10) (Figure 3A, inset). This response increased with time, reaching a maximum after approximately 2 min. The depolarization produced by IGF-I (100 ng/ml) was not significantly changed by pre-incubating the Sertoli cells with PTX (1 μg/ml) for 60 min (Figure 3A). However, IGF-I-induced depolarization was inhibited in the presence of verapamil (100 μM) (an inhibitor of L-type VDCC) at all times studied (**p < 0.001; n = 6) (Figure 3B).

FIGURE 3

Figure 3. Rapid depolarization of immature Sertoli cells produced by topical application of IGF-I (25 ng/ml) (A, inset). The IGF-I depolarization time course was not affected by pre-incubation with PTX (1 μg/ml) (A), but was nullified by the presence of verapamil (100 μM) (B). Sertoli cells were pre-incubated with PTX for 1 h before IGF-I application, and verapamil was perfused 10 min before IGF-I application. *p < 0.05; n = 5 in each time from five independent experiments; **p < 0.001; n = 6 in each treatment (IGF-I or IGF-I plus verapamil) from five separated experiments.

FSH-Induced Stimulation of ⁴⁵Ca²⁺ Entry

Follicle stimulating hormone stimulated ⁴⁵Ca²⁺ uptake within 2 min of its addition to testes. This stimulatory effect of FSH on ⁴⁵Ca²⁺ uptake was blocked by 60 min pre-incubation with PTX (Figure 4A), suggesting that the rapid effect of FSH is mediated by Gi proteins. FSH-stimulated ⁴⁵Ca²⁺ entry was also inhibited by 60 min pre-incubation with wortmannin (Figure 4B), indicating that the PI3K pathway is also required for this action of the hormone. Pre-incubation for 10 min with verapamil also inhibited FSH stimulation of ⁴⁵Ca²⁺ entry (Figure 4D). However, stimulation of AC by forskolin (100 nM) during the 2 min of incubation did not change ⁴⁵Ca²⁺ uptake in immature rat testes (Figure 4C).

FIGURE 4

Figure 4. Follicle stimulating hormone produces an increase in ⁴⁵Ca²⁺ uptake within 2 min of incubation. This increase was inhibited by PTX (A), wortmannin (B), and verapamil (D). Forskolin was not able to increase ⁴⁵Ca²⁺ in 2 min (C). (A)*p < 0.05, as compared with the other groups, n in brackets from two separated experiments; (B,D) *p < 0.05, as compared with the other groups, n = 5 from one experiment.

FSH-Stimulated [¹⁴C]-MeAIB Transport was Inhibited by PTX and Wortmannin

Follicle stimulating hormone, through the increase of calcium uptake and the consequent stimulation of microtubules activity (Wassermann et al., 1992a), produces a fast increase (seconds) (Silva et al., 2002) in amino acid transport in Sertoli cells during the proliferative phase (between 10- to 22-day-old rats) (Pérez-Sánches and Wassermann, 1981). The stimulatory effect of FSH (4 mU/ml) on the transport of the model amino acid [¹⁴C]-MeAIB was abolished by the pre-incubation with PTX (Figure 5A) (*p < 0.05; n = 5). This stimulatory action of FSH was also blocked by wortmannin, an inhibitor of PI3K (Figure 5B) (*p < 0.05; n = 5).

FIGURE 5

Figure 5. Follicle stimulating hormone stimulation of [¹⁴C]-MeAIB transport was blocked by PTX (1 μg/ml) (A) and by wortmannin (100 nM). (B) *p < 0.05, as compared with the other groups, n = 5 from one experiment.

Actions of IGF-I On ⁴⁵Ca²⁺ uptake and [¹⁴C]-MeAIB Transport in Immature Sertoli Cells

The incubation of immature rat testes with IGF-I (25 ng/ml) for 2 min increased ⁴⁵Ca²⁺ uptake (*p < 0.05; n = 9) (Figure 6A). A similar stimulatory action of IGF-I was observed using [¹⁴C]-MeAIB in testes incubated for 45 min with different concentrations of the growth factor (Figure 6B). The stimulatory action of IGF-I on [¹⁴C]-MeAIB was also observed in testes pre-incubated for 60 min with PTX (250 ng/ml) (Figure 6C). This result indicates that, unlike FSH, the stimulatory effect of IGF-I on [¹⁴C]-MeAIB transport was not affected by the presence of PTX (Figure 6C) (*p < 0.05; n = 5 as compared with the other groups), suggesting that IGF-I and FSH utilize different signaling pathways to elicit this response.

FIGURE 6

Figure 6. IGF-I (25 ng/ml) increases ⁴⁵Ca²⁺ uptake within 2 min of incubation (A) and [¹⁴C]-MeAIB transport at different doses of IGF-I (B). PTX (250 ng/ml) did not affect [¹⁴C]-MeAIB transport stimulated by IGF-I (100 ng/ml) (C). Samples were pre-incubated for 60 min with PTX and incubated for 45 min with [¹⁴C]-MeAIB and IGF-I. Statistical analysis: a Student’s t-test was performed in (A) (*p < 0.05; n in brackets from two independent experiments); a two-way ANOVA followed by Bonferroni post test. (B) *p < 0.05; n = 5 in each concentration from five separated experiments, (C) *p < 0.05 as compared with the other groups, n = 5 from one experiment.

Effect of the IGF-I Receptor Blocking Agent, JB1, on the Actions of IGF-I and FSH

The stimulatory effect of IGF-I (25 ng/ml) on ⁴⁵Ca²⁺ entry was inhibited by JB1 (1 μg/ml), an inactive analog of IGF-I (Figure 7A). However, this analog had no effect when applied in combination with FSH (4 mU/ml) (Figure 7B) (*p < 0.05; n = 5 as compared with the other groups).

FIGURE 7

Figure 7. Effect of the inactive analog of IGF-I, JB1 (1 μg/ml), on IGF-I (A) and FSH (B) stimulation of ⁴⁵Ca²⁺ entry after 2 min of incubation. JB1 inhibited the stimulatory effect of IGF-I on ⁴⁵Ca²⁺ entry, but not the action of FSH, on ⁴⁵Ca²⁺ uptake. (A) *p < 0.05 as compared with the other groups, n in brackets from two independent experiments; (B) *p < 0.05 as compared with the other groups, n in brackets from three separated experiments.

Effect of an anti-IGF-I Antibody on the Actions of IGF-I and FSH on Membrane Potential and [¹⁴C]-MeAIB Transport

Anti-IGF-I antibody (500 ng/ml) abolished the depolarization caused by IGF-I (100 ng/ml) (Figure 8A), but did not interfere with the typical MP changes induced by FSH (Figure 8B). Similarly, the anti-IGF-I antibody inhibited IGF-I-stimulated [¹⁴C]-MeAIB transport at all concentrations tested (Figure 8C), but did not inhibit the FSH response (Figure 8D) (*p < 0.05; n = 5 as compared with the other groups).

FIGURE 8

Figure 8. Effect of anti-IGF-I antibody (500 ng/ml) on the action of IGF-I and FSH on membrane potential and [¹⁴C]-MeAIB transport. The anti-IGF-I antibody inhibited the depolarization produced by IGF-I on MP (A) but it did not interfere with the MP modifications produced by FSH (B). The antibody inhibited the action of IGF-I on [¹⁴C]-MeAIB transport at all concentrations tested (C) but did not inhibit the effect of FSH (D). Sertoli cells were pre-incubated with anti-IGF-I antibody for 1 h before IGF-I or FSH application. (A) **p < 0.001; n = 5 in each treatment (IGF-I or IGF-I plus anti-IGF-I) from five separated experiments; (C) *p < 0.05; n = 5 or in brackets, one separated experiments for each concentration; (D) *p < 0.05; n in brackets, two separated experiments for each concentration.

Discussion

The aim of this study was to evaluate the effect of PTX on the depolarizing phase of FSH stimulation on the MP of Sertoli cells from 10- to 12-day-old rats, which is linked to the rapid entry of Ca²⁺ (Wassermann et al., 1992b; Loss et al., 2007) and the Ca²⁺-dependent transport of neutral amino acids by the A system (Wassermann et al., 1992a). In these experiments we analyzed the involvement of Gi proteins in FSH-induced effects. In parallel, we investigated the possible mediating role of IGF-I on the effects of FSH, as IGF-I is produced and released by testicular cells in response to FSH stimulation (Khan et al., 2002) and has a depolarization effect on the MP of Sertoli cells that is similar to FSH (Jacobus et al., 2010).

We found that the rapid membrane actions of FSH that occur in a time-frame of seconds to minutes, including membrane depolarization, stimulation of ⁴⁵Ca²⁺ uptake and [¹⁴C]-MeAIB transport, were prevented by PTX and, therefore, it is likely that these effects are mediated by GiPCR activation. Furthermore, these effects were also inhibited by verapamil, a blocker of the L-type VDCC. Finally, FSH stimulation of ⁴⁵Ca²⁺ uptake and [¹⁴C]-MeAIB transport were inhibited by wortmannin, an inhibitor of PI3K. These FSH effects were independent of IGF-I.

In earlier studies, Grasso and Reichert Jr. (1989) observed a suppression of FSH-stimulated ⁴⁵Ca²⁺ uptake by methoxy-verapamil and nifedipine, specific blockers of voltage-activated calcium channels. These authors also reported that FSH stimulates ⁴⁵Ca²⁺ entry within 1 min (surge phase) (Grasso et al., 1992). In another experiment, after 72 h in culture, cells were pre-incubated (4 h) with PTX and then incubated for 24 h with FSH plus 0.4 μCi of ⁴⁵Ca²⁺. Then, the cells were washed and ⁴⁵Ca²⁺ was measured. In this experiment, PTX was ineffective at blocking the effect of FSH. However, this experimental model is suitable for studying the sustained phase of FSH action, which is probably mediated by cAMP, but not the surge phase of ⁴⁵Ca²⁺ entry (Figure 6B from Grasso and Reichert, 1990). In a supplementary experiment, the authors incubated with FSH for 2 min and added ⁴⁵Ca²⁺ at the same time (Table 4 from Grasso and Reichert, 1990), but did not observe an effect of PTX under these experimental conditions.

The results of PTX treatment in isolated Sertoli cells by Gorczynska et al. (1994) and in our experiments (Figures 1C, 4A and 5A) conflict with this last result of Grasso and Reichert (1990). Other than the differences in time and treatment methods used, as described, the reason for this discrepancy is not entirely clear, but may be due to differences in the experimental approach or technical procedures. In our ⁴⁵Ca²⁺ experiments, we pre-incubated with ⁴⁵Ca²⁺ until an intracellular plateau was reached, after approximately 60 min. At that time, we added FSH (4 mU/ml) and incubated the samples for an additional 2 min. At the end of the incubations, we were very careful to avoid losing intracellular ⁴⁵Ca²⁺ during washes, using LaCl₃ in the washing solutions (Grasso and Reichert Jr., 1994; Batra and Sjogren, 1983; Grasso et al., 1992; ).

Using a different approach, measuring cytoplasmic Ca²⁺ through a Fura2-fluorescence technique, Sharma et al. (1994) observed that the immediate FSH-induced rise in [Ca²⁺]i was due to Ca²⁺ entry. Additionally, they showed that verapamil and Co²⁺ both abolished the FSH-triggered increase in [Ca²⁺]i. Furthermore, Ca²⁺-free medium prevented this FSH effect. They suggested that PTX does not affect the FSH-induced [Ca²⁺]i rise. However, this interpretation is based on only one representative record (Figure 7A from Sharma et al., 1994) without statistical processing, which they did carry out for the PTX-potentiated FSH-stimulated accumulation of cAMP (Figure 7B from Sharma et al., 1994).

In the absence of extracellular Ca²⁺, cAMP analog (8-Br-cAMP and dbcAMP) also induce a rise in [Ca²⁺]i, which is probably due to the mobilization of Ca²⁺ from internal stores (Sharma et al., 1994) in the cAMP-dependent sustained phase (Grasso et al., 1992). Gorczynska et al. (1994) also reported differences between the FSH- and cAMP-mediated cytosolic increases in Ca²⁺. They found that verapamil abolished the FSH-stimulated transmembrane Ca²⁺ flux (surge phase) in Sertoli cells, but a calcium channel-blocking agent or the absence of extracellular Ca²⁺ only partially blocked the cytosolic increase produced by cAMP, indicating an intracellular source of calcium for this increase (sustained phase). Lalevée et al. (1999) reported that the peak of calcium induced by cAMP was suppressed in Sertoli cells exposed to thapsigargin, suggesting the release of calcium from smooth endoplasmic reticulum. The relationship between cAMP and calcium following FSH stimulation is clearly complex. Differences in the origin of calcium in the FSH- versus cAMP-induced cytosolic calcium increase have been observed. The FSH-stimulated cytosolic calcium rise in freshly isolated Sertoli cells is dependent on the availability of extracellular calcium (Gorczynska and Handelsman, 1991), but a large proportion of the calcium participating in the cAMP-induced calcium rise is derived from the intracellular calcium sources (Gorczynska et al., 1994). These results indicate that a cAMP-independent pathway for increasing cytosolic calcium might be used by Sertoli cells. Therefore, the physiological responses of Sertoli cells to FSH might be dependent on the continuous cross-talk of both cAMP-dependent and cAMP-independent pathways (Gorczynska-Fjalling, 2004).

The average resting potential of seminiferous tubules in our experiments on 10- to 12-day-old rats was –44 ± 1.1 mV (n = 73), and the membrane input resistance was 10 ± 0.5 MΩ (n = 73). The basal electrical characteristics in seminiferous tubules of 12 and the membrane input resistance 9.3 ± 0.7 MΩ (n = 30). To avoid working with germ cells, only cells with MP lower than −35 mV were included in the experiments, as this MP is commonly found in Sertoli cells from normal (Eusebi et al., 1983) or Sertoli cell enriched seminiferous tubules of pre-pubertal rats (Wassermann et al., 1992b). The tip resistance that we used (15–25 MΩ) was appropriate for the preferential impalement of cells with a size similar to that of Sertoli cells (von Ledebur et al., 2002). This tip diameter makes the impalement of smaller cells, such as peritubular myoid cells, difficult. The membrane input resistance in Sertoli cells from normal seminiferous tubules is relatively lower than that found in cells from Sertoli cells-enriched seminiferous tubules (Eusebi et al., 1983) or isolated Sertoli cells (50 ± 5.2 MΩ; n = 10) (data not shown). The low membrane resistance is probably due to the fact that Sertoli cells in normal seminiferous epithelia are electrically coupled to each other and to germ cells. To preserve the gap junctions of immature Sertoli cells, whole seminiferous tubules were used in the present work, which meant that normal functional integrity of the environment was preserved as well as possible (Eusebi et al., 1983; von Ledebur et al., 2002).

In our experimental conditions, topical application of FSH produced changes in the MP of Sertoli cells. An immediate hyperpolarization was followed by depolarization (more than 6 min), returning to the resting potential after this period (Figure 1B). The depolarizing response was blocked by pre-treatment with verapamil (Wassermann et al., 1992b), indicating the involvement of L-type VDCC on the action of FSH.

Gorczynska and Handelsman (1991) reported that exchanging sodium and potassium concentrations in the extracellular medium (high potassium and low sodium, an ionic solution that produces MP depolarization) led to an immediate increase in cytosolic calcium, and subsequent FSH administration did not further increase these levels. The absence of external calcium in the incubation medium abolished the initial rise in the cytosolic free calcium concentration that was induced by FSH.

The gating of L-type calcium channels can be regulated by the α1 subunit of the channel through voltage changes in the cellular membrane, i.e., voltage clamp or ionic manipulation (Gorczynska and Handelsman, 1991), or by specific phosphorylation of the β subunit by PKB (Dolphin, 2003; Viard et al., 2004).

Phosphoinositide 3-kinases are enzymes that catalyze the phosphorylation of membrane phosphoinositides at the inositol 3-OH position. Class I, receptor-regulated PI3Ks connect many extracellular stimuli to intracellular responses. Class I PI3Ks are subdivided into class 1A (PI3K α, β, δ) kinases, which are activated by tyrosine kinase receptors (TKRs) or non-receptor tyrosine kinases, and the class 1B (PI3Kγ), which is uniquely activated by GPCRs and regulated by free Gβγ subunits of heterotrimeric G proteins, usually of the Gi subtype (Hirsch et al., 2007; Musnier et al., 2010). The PI3K downstream pathway leads to PIP2 phosphorylation producing PIP3, and this phosphoinositide then enhances PKB/Akt activation (Hirsch et al., 2007).

Active PKB/Akt is able to phosphorylate a vast number of proteins, thus representing a key effector of PI3K signaling. One of the actions reported for PKB/Akt is the control of extracellular Ca²⁺ entry due to a direct effect on the L-type Ca²⁺ channel leading to its plasma membrane exposure (Viard et al., 2004). These data allow us to speculate that PI3Kγ, activated by the βγ subunits of Gi proteins, could mediate the FSH stimulation of L-type calcium channels. Despite the fact that the molecular mechanism of this hypothetical stimulation is not clear, it could be achieved through PIP3 generation by activated PI3Kγ isoforms, which in turn could activate the β-subunit (regulatory unit) of the L-type Ca²⁺ channels through PKB, as was suggested by Viard et al. (2004) for adrenergic receptors, and by Le Blanc et al. (2004) for angiotensin II receptors in vascular cells.

It has been also reported that FSH produces a fast enhancement (seconds) in the activity (V_max) of functional transporters of neutral amino acids in the membrane of Sertoli cells (Silva and Wassermann, 1999; Silva et al., 2002) through the increase of Ca²⁺ uptake and consequent stimulation of microtubule activity (Wassermann et al., 1992a). This FSH stimulatory action was independent of its effect on AC-cAMP activity and was observed using FSH from different species and sources (NHPP: ovine, porcine, and rat; Sigma-Aldrich: ovine) (Irusta and Wassermann, 1974; Pérez-Sánches and Wassermann, 1981; Cruz Curte and Wassermann, 1985; Spritzer and Wassermann, 1985; Silva and Wassermann, 1999; Silva et al., 2002).

The stimulatory action of the IGF-I on [¹⁴C]-MeAIB transport observed in immature Sertoli cells (Figures 6A,B) is probably processed through similar Ca²⁺-dependent mechanism activated by class 1A (PI3K) kinases (Figure 9). In addition to the complex role of IGF-I signaling in pre-pubertal testes, studies in neuronal cells have demonstrated that PTX inhibits IGF-I-mediated activation of MAPK, with a specific role for Gβγ subunits in IGF-I signaling (Hallak et al., 2000). These results show an association of heterotrimeric Gi with the IGF-I receptor. Inhibitory effects of PTX have also been reported on other cellular actions of IGF-I (Jin et al., 1993; Poiraudeau et al., 1997; Sarbassov et al., 1997; Uehara et al., 1999), but this was not seen in all cases (Stracke et al., 1988; Langlois et al., 1990; Linder et al., 1994; Sieble et al., 1996). Thus, in different cell types, Gβγ subunits can mediate cross-talk between GPCR and TKR signaling. However, this interrelationship can be discounted for the effects studied here in Sertoli cells, as PTX did not inhibit or modify the IGF-I depolarization of the MP (Figure 3A) or stimulation of [¹⁴C]-MeAIB transport (Figure 6C), unlike FSH (Figure 5A).

FIGURE 9

Figure 9. Hypothetical model of FSH action on Ca²⁺ uptake and amino acids transport. Numbers indicate pharmacological agent effects on different steps of the FSH action: 1 – PTX blocks Gi protein; 2 – Wortmannin blocks PI3K; 3 – Verapamil inhibits Ca²⁺ uptake and amino acid transport; 4 – JB1 and anti-IGF-I antibody block IGF-I action but not the FSH action; 5 – tolbutamide inhibits the channel; 6 – Forskolin, that activates adenylate cyclase, produces hyperpolarization and did not affect Ca²⁺ uptake. VDCC, voltage-dependent Ca²⁺ channel (surge phase); Na⁺-aa, neutral amino acid co-transport; ER, endoplasmic reticulum (sustained phase).

Because of the close relationship between FSH and IGF-I in immature testis (Loss et al., 2007), we examined the effects of an anti-IGF-I antibody and an inhibitory analog of IGF-I, JB1, on the effects of FSH and IGF-I. The anti-IGF-I antibody abolished the depolarization caused by IGF-I (Figure 8A) but did not interfere with the typical MP modification produced by FSH (Figure 8B). Similarly, the anti-IGF-I antibody inhibited IGF-I-stimulated [¹⁴C]-MeAIB transport at all concentrations tested (Figure 8C), but it was ineffective in the inhibition of FSH activity (Figure 8D). The same effects on ⁴⁵Ca²⁺ uptake were observed using JB1. These results indicate that the effects of FSH that we observed were unrelated to IGF-I.

In summary, the calcium related rapid effects of FSH on Sertoli cell membranes from 10- to 12-day-old rats were blocked by specific pharmacological inhibitors: PTX (250 ng/ml, 1000 ng/ml), wortmannin (100 nM), and verapamil (100 μM). These results suggest the following pathway for the rapid action of FSH on Sertoli cell membranes: Gi protein activates PI3Kγ through its βγ subunits, by means of an unknown mechanism (most likely through PIP3-activated PKB/Akt (Viard et al., 2004; Vecchione et al., 2005; Hirsch et al., 2007), and stimulates extracellular ⁴⁵Ca²⁺ entry (current) through the voltage-dependent L-type Ca²⁺ channel, which depolarizes the membrane and activates the A system for neutral amino acid transport (Figure 9). This hypothesis deserves further investigation to clarify the different steps involved in this pathway.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by grants from CNPq. The authors are indebted to Prof. Luis Carlos Kusharski for the generous supply of ⁴⁵Ca²⁺ and Dr. A. Parlow from the National Hormone and Peptide Program (NHPP), National Institute of Diabetes and Digestive and Kidney Disease (NIDDKD) at the UCLA Medical Center (Torrance, CA, USA) for the drug contribution.

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