Angiotensin II Stimulates Contraction and Growth of Testicular ...

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Endocrinology 143(8):3096 –3104 Copyright © 2002 by The Endocrine Society

Angiotensin II Stimulates Contraction and Growth of Testicular Peritubular Myoid Cells in Vitro F. ROSSI, A. FERRARESI, P. ROMAGNI, L. SILVESTRONI,

AND

V. SANTIEMMA

Dipartimento di Fisiopatologia Medica, Universita` di Roma “La Sapienza” Facolta` di Medicina, 00161 Rome, Italy Seminiferous tubule contraction, an important step in the regulation of spermatogenesis and testicular sperm output, is regulated by several agonists. In the present paper, we investigated whether angiotensin II (Ang II) may have a place among them. In binding experiments performed to assess the presence of specific receptors in rat peritubular myoid cells (TPMC), binding of 125I-Ang II to TPMC was saturable in a time-dependent manner. Competition binding experiments performed with Losartan and PD 123319 showed that Losartan was able to inhibit the binding of 125I-Ang II, whereas PD 123319 was ineffective. Ang II induced a dose-dependent rise in intracellular Ca2ⴙ. Depletion of intracellular calcium stores by thapsygargin resulted in a lower rise of intracellular calcium, and the L-type voltage-operated calcium channel (VOCC-L) blocker verapamil abolished the Ca2ⴙ influx in rat TPMC. Altogether, these findings indicate that the Ang II-induced increase in [Ca2ⴙ]i involves both extracellular influx and Ca2ⴙ release from intracellular stores.

T

ESTICULAR PERITUBULAR MYOID cells (TPMCs), the main cellular component of the seminiferous tubule wall, take part in the paracrine regulation of testis function. In vitro studies have demonstrated that TPMCs secrete a number of substances, including extracellular matrix components (fibronectin, type I and IV collagens, proteoglycans), and growth factors [peritubular factor that modulates Sertoli cell function (PModS), TGF-␤, IGF-I, activin-A], some of which are known to enhance Sertoli cell secretion in a paracrine fashion (1). TPMC, as contractile cells, express the cytoskeletal markers of true smooth muscle cells (SMC), such as ␣-isoactin, F-actin, and myosin. The arrangement of the actin filaments in TPMC changes during postnatal development and seems to be further affected by the disruption of spermatogenesis (e.g. in cryptorchidism). In the rat, the filaments within one myoid cell run both longitudinally and circularly to the long axis of the seminiferous tubule, and their contractile activity is responsible for the contraction of seminiferous tubules involved in the transport of spermatozoa and testicular fluid, and at least partially, for sperm release during spermiation (2, 3). Several agonists (endothelin-1, arginine vasopressin, TGF␤, platelet-derived growth factor, oxytocin, and prostaglandins) affect TPMC contraction in endocrine, paracrine, and autocrine fashion (4 –7). Abbreviations: AM, Adrenomedullin; AngII, angiotensin II; AT1 and -2, angiotensin II receptor type 1 and 2, respectively; FCS, fetal calf serum; MEK, MAPK kinase; PKC, protein kinase C; RAS, renin-angiotensin system; SMC, smooth muscle cell; TPMC, testicular peritubular myoid cell; VOCC-L, L-type voltage-operated calcium channel.

Ang II induced a dose-dependent TPMC contraction, and Losartan and not PD 123319 inhibited the response. Ang IIinduced contraction was inhibited by adrenomedullin, previously shown to antagonize endothelin 1-provoked contraction in those cells. Ang II elicited 3H-thymidine DNA incorporation and proliferation in a dose-dependent manner in TPMC. Losartan and both MAPK inhibitor PD 98059 and tyrosine kinase inhibitor AG18 were able to inhibit Ang II-induced 3H-thymidine uptake and cell proliferation. In conclusion, the present study documents that angiotensin II, the active mediator of the tissue and circulating reninangiotensin system present in the mammalian testis, induces contraction, growth and rise in intracellular calcium in rat peritubular myoid cells via angiotensin II type 1 receptors, and suggests that Ang II is involved in the paracrine regulation of the seminiferous tubule function. (Endocrinology 143: 3096 –3104, 2002)

All this being considered, it is evident that peritubular myoid cells not only provide structural integrity to the tubule but also take part in the regulation of spermatogenesis and overall testicular function. Ang II is a multifunctional hormone, the active mediator of the tissue and circulating renin-angiotensin system (8), that exerts a wide variety of physiological effects. The role of systemic renin-angiotensin system in cardiovascular function, including contraction and proliferation of vascular and visceral SMCs, and electrolyte homeostasis has been well documented (9, 10). Local variants have been identified and postulated to be involved in autocrine and/or paracrine mechanisms at tissue level (11–13). Angiotensin II affects growth and differentiation of its target tissues and elicits contraction in several SMC types via AT1 receptors (14). The presence of all the components of renin-angiotensin system has been recently reported in the testis (15–17), and a kinase type II (angiotensin converting enzyme) testisspecific isozyme is present in the seminiferous epithelium of the rat (18). The epididymis also contains angiotensin II receptors (19 –21) and somatic ACE (22). Moreover, in the seminiferous tubules of Sprague-Dawley rats, Ang II, and AT1 and AT2 receptors are present at 3 wk of age. In the interstitial space, both AT1 and AT2 receptors are present at 10 d of age, whereas at 7 wk only the type 1 receptor is present (23–25). In the present study, we investigated whether Ang II binds and can affect intracellular Ca2⫹, contraction, and proliferation in testicular peritubular myoid cells, thereby qualifying

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FIG. 1. Competitive binding of 125I-Ang II to testicular peritubular myoid cells. Competition binding experiments were performed using 0.2 nM 125IAng II in the presence of increasing concentration of unlabelled Ang II at 22 C. The values are expressed as 125I-Ang II bound %. Each point is expressed as the mean ⫾ SEM of four separate experiments run in duplicate. A representative Scatchard analysis is shown in the inset.

as a new agonist in the paracrine regulation of the seminiferous tubule. Materials and Methods Materials Endothelin-1, angiotensin II, and PD 125319 were obtained by Sigma (Milan, Italy). Losartan was kindly provided by Merck, Sharp & Dohme (Rome, Italy). PD 98059 and AG18 were purchased from Calbiochem (San Diego, CA). Adrenomedullin (AM) was purchased from Peptide Institute (Osaka, Japan). 125I-Ang (2000 Ci/mmol) and 3H-thymidine (84 Ci/mmol) were obtained from Amersham Pharmacia Biotech (Milan, Italy).

Rat TPMC isolation and culture Rat TPMC were isolated, as previously described (7), from 18-d-old Sprague Dawley rats provided by Charles River (Calco, Italy). Primary cultures of rat TPMC were maintained at 32 C in 5% CO2 atmosphere in DMEM/F12 (1:1) containing 10% fetal calf serum (FCS; Life Technologies, Inc., Grand Island, NY), and the medium was changed every 48 h.

Binding study TPMC cultured in 24-well plates were washed twice in Dulbecco’s PBS (Life Technologies, Inc.) containing 0.2% BSA, 5 mm glucose, 10 mm MgCl2, and the protease inhibitor phenylmethylsulfonyl fluoride (0.1 mm). To evaluate specific binding, saturation binding experiments were performed at 22 C using increasing concentrations of 125I-Ang II in the absence (total binding) and in the presence (nonspecific binding) of 1 ␮m unlabeled Ang II. Competition binding experiments with Ang II, Losartan, and PD 123319 were performed using 0.2 nm 125I-Ang II and increasing concentrations of the indicated compounds. At the end of the incubation, the cells were washed three times with cold Dulbecco’s PBS containing 0.2% BSA, solubilized in 0.1 n NaOH, and counted with a ␥ counter at an efficiency of 70%. Nonspecific binding averaged 10 –15% of total binding.

FIG. 2. Competitive binding of 125I-AngII (0.2 nM) to testicular peritubular myoid cells in the presence of increasing concentration of unlabelled Ang II (䡺), Losartan (E) or PD 123319 (F). Losartan showed a capacity to compete with the binding of 125I-Ang II with Ki 1.03 ⫻ 10⫺7 M. The values are expressed as 125I-Ang II bound %. Each point is expressed as the mean ⫾ SEM of four separate experiments run in duplicate.

Measurement of [Ca2⫹]i For the Ca2⫹ measurements we used a SIM buffer containing (in mm) 30 HEPES, 5 KCl, 1.2 MgSO4, 5.5 glucose, 122 NaCl, 1.5 CaCl2 and NaOH to pH 7.4. In Ca2⫹-free experiments, the same solution was used except that CaCl2 was omitted and 0.5 mm EGTA was added. [Ca2⫹]i was measured by dual wavelength fluorescence in single cells loaded with the Ca2⫹-sensitive indicator fura-2 by spectrofluorometry (SFM 25, Kon-

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FIG. 3. Pattern of [Ca2⫹]i in fura 2-loaded testicular peritubular myoid cells in response to angiotensin II (100 nM) in the presence of extracellular calcium (A) and in calcium-free medium (B). The effect of two subsequent stimulations with Ang II is shown in C, and the effect of ET1 (100 nM) after a first stimulation with Ang II is shown in D. For the Ca2⫹ measurements, we used the SIM buffer as in Materials and Methods. In Ca2⫹-free experiments, the same solution was used except that CaCl2 was omitted, and 0.5 mM EGTA was added. [Ca2⫹]i was measured by dual wavelength fluorescence in single cells loaded with fura-2 by spectrofluorometry with 340 nm of excitation and 505 nm of emission. The traces are representative of at least four consistent experiments. tron Instruments Ltd., Watford, UK) with 340 nm of excitation and 505 nm of emission. The maximum fluorescence was assessed after cells permeabilization by 2 ␮m ionomycin, and the minimum fluorescence after addition of 1 mm MnCl2 according to the formula:

Ca⫹⫹(nM) ⫽ Kd ⫻ (F ⫺ Fmin)/(Fmax ⫺ F)

TPMC microperfusion chamber system The microperfusion experiments were performed, as already described (26). In brief, a glass coverslip with plated testicular peritubular myoid cells was placed into a stainless steel microperfusion chamber whose volume was 0.5 ml. Perfusate was circulated by a peristaltic pump. A multiport valve served to switch between perfusates with different composition. Control of the temperature inside the cell chamber (37 C) was achieved by external control of perfusate temperature. Measurements were performed upon continuous cell perfusion on an Olympus microscope (Olympus,

Tokyo, Japan) using a 20⫻ phase contrast objective. Images were video captured by a Panasonic (Milan, Italy) B/W camera coupled to a Sony (Milan, Italy) video recorder. After perfusion (1 ml/min) with regular buffer for 3 min, time necessary for thermal equilibration, cells were flushed with test compounds. To evaluate cell contraction, the videocaptured images were acquired on computer (Screen Machine II, FAST Multimedia AG, Munchen, Germany), cell areas were calculated from the digitized images (SigmaScan Pro, Jandel Scientific, Erkrath, Germany), and percent of contraction (C) computed according to the formula:

C ⫽ [(A ⫺ a)/A] ⫻ 100 where A is the area of a single cell before stimulation and a is the area of the same cell after stimulation.



FIG. 4. The decrease in fluorescence intensity to levels below control levels after the addition of Ang II, when 50 nM Mn2⫹ was substituted for extracellular Ca2⫹, indicates the opening of membrane calcium channels (A). Pattern of [Ca2⫹]i in fura 2-loaded testicular peritubular myoid cells in response to Ang II (100 nM) in Ca2⫹-free medium after depletion of intracellular calcium stores by thapsigargin (B). Effect of L-type voltage-operated calcium channel (VOCC-L) blocker Verapamil (5 ␮M) on Ang II-induced rise in [Ca2⫹]i (C). The traces are representative of at least four consistent experiments.

Incorporation of 3H-thymidine TPMC were subcultured and plated in 24-well plates (Falcon Plastic, Los Angeles, CA) at a density of 1 ⫻ 105 cells/well and cultured overnight in DMEM containing 10% FCS at 34 C. Cell cycle synchronization was performed in serum-free medium for 24 h before 3H-thymidine assays. After administration of Ang II, ET1, AM, or no treatment for 20 h, cells were incubated with 3H-thymidine (1 ␮Ci/ml) for 4 h. The cells were then washed three times with PBS and precipitated twice for 5 min with 10% trichloroacetic acid. After aspiration of supernatant, the precipitate was solubilized in 0.5 ml 1 n NaOH and neutralized with 0.5 n HCl. The tritium incorporation was determined by scintillation counting. To investigate the specificity of the mitogenic

effect of Ang II, the cells were preincubated, adding compound to cell culture 20 min before Ang II treatment with 100 nm Losartan, AT1 selective antagonist, or 100 nm PD 123319, AT2 selective antagonist. MAPK and tyrosine kinase involvement in the mitogenic effect of Ang II was determinated by adding the MAPK inhibitor PD 98059 (5 ␮m) and the tyrosine kinase inhibitor AG18 (1 ␮m). Experiments were performed in triplicate.

Cell growth analysis To evaluate proliferation, subconfluent testicular peritubular myoid cells were subcultured in 24-well plates (Falcon Plastic, Los Angeles, CA) at a density of 1 ⫻ 105 cells/well and cultured overnight in DMEM

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FIG. 5. Dose-response curve of testicular peritubular myoid cells contraction induced by graded concentration (2 ⫻ 10⫺9–10⫺6 M) of Ang II (EC50 10⫺9 M) (F). Testicular peritubular myoid cells were plated on a glass coverslip that was placed into a stainless steel temperaturecontrolled (37 ⫾ 0.2 C) microperfusion chamber. Contraction was evaluated through image analysis as described in Materials and Methods. Pretreatment with Losartan (100 nM) (E) produced a rightward parallel shift in the dose-response curve for Ang II without affecting the maximal response. Schild analysis of the data gave a pA2 value of 8.156 and a slope of 0.99. The results are expressed as percentage of contraction computed as the difference in the areas before and after stimulation. Student’s paired t test was used for the statistical analisys of data. Each point is expressed as the mean of three separate experiments run in triplicate. Bars show SEM. containing 10% FCS at 34 C. Then cells were made quiescent by incubation for 24 h in serum-free medium. Adherent cells, cultured in DMEM containing 0.3% FCS, were treated with graded concentration of Ang II (10⫺9–10⫺6 m) or with drug solvent only. After 24 h, Ang II was added again to cell culture. After a further 24 h, cells were rinsed with PBS and removed by trypsin/EDTA treatment for 7 min, centrifuged at 900 rpm for 15 min, and resuspended in 1 ml of DMEM for counting in a cell counter chamber. Cell viability was determined by trypan blue dye exclusion. When peptide antagonists, tyrosine kinase inhibitor, or MAPK inhibitor were used, agents were added to cell culture 20 min before Ang II treatment. For each treatment, four wells were counted in triplicate, and experiments were carried out on three separate cultures.

Statistical analysis Data are presented as means ⫾ sem. Statistical analysis was performed by using the Student’s paired t test for cell contraction experiments and Student’s unpaired t test for all the other experiments. P ⬍ 0.05 was taken as statistically significant.

Binding of

125

Results I-Ang II to TPMC

Binding of 125I-Ang II to TPMC was saturable in a timedependent manner, and specific binding was maximal at 60 min (data not shown). Scatchard analysis from saturation experiments showed a single class of binding sites (dissociation constant of 4.5 ⫾ 0.5 nm, and maximal binding capacity


FIG. 6. Effect of graded concentration (10⫺10–10⫺5 M) of adrenomedullin on 100 nM Ang II-induced contraction. AM inhibited contraction in dose-dependent fashion with an IC50 of 2 ⫻ 10⫺8 M. The results are expressed as percentage of contraction computed as the difference in the areas before and after stimulation. Each point is expressed as the mean ⫾ SEM of three separate experiments run in triplicate performed in the microperfusion chamber system as above.

of 1912 ⫾ 672.3 fmol/105 cells) (Fig. 1). Competition binding experiments were performed with 125I-Ang II, unlabeled Ang II, Losartan, and PD 123319. Losartan showed a capacity to compete with the binding of 125I-Ang II with Ki (constant of inhibition) 1.03 ⫻ 10⫺7 m, whereas no changes were observed in the presence of PD 123319 (Fig. 2), suggesting that TPMC express AT1 receptors. Effect of Ang II on [Ca2⫹]i

Angiotensin II (100 nm) induced a rapid increase in fura-2 fluorescence (Fig. 3A). After the first stimulation, further addition of Ang II did not elicit a response in [Ca2⫹]i suggesting a typical desensitization (Fig. 3C). However, if ET1, instead of Ang II, was added as the second agonist, elevation in [Ca2⫹]i was still promoted (Fig. 3D). As in Ca2⫹-free experiments Ang II stimulation resulted in increased [Ca2⫹]i (Fig. 3B), which suggested mobilization of intracellular calcium, we evaluated a putative mechanism of Ca2⫹ mobilization from internal stores. In the presence of 50 nm Mn2⫹ (Fig. 4A), Ang II addition resulted first in increased fura-2 fluorescence— due to intracellular calcium mobilization—and then in lower than baseline fura-2 fluorescence— due to quenching by Mn2⫹, which enters the cell via calcium channels. All together, this indicates that Ang II induced both extracellular calcium influx and Ca2⫹ release from intracellular stores. Depletion of intracellular calcium stores by thapsygargin (Fig. 4B) before Ang II addition, in the absence of extracel-



FIG. 7. Testicular peritubular myoid cell contraction induced by ET1 (100 nM) in the absence or in the presence of specific antagonist BQ123 (100 nM) and by Ang II (100 nM) in the absence or in the presence of Losartan (100 nM) or PKC inhibitor calphostin (100 nM). The results are expressed as percentage of contraction. Data are normalized assuming as 100% the maximal response evoked by ET1 (100 nM). Each histogram represents the mean ⫾ SEM of three separate experiments run in triplicate. **, Significant difference (P ⬍ 0.001) vs. samples treated with angiotensin II only.

lular Ca2⫹, completely abolished the rise of intracellular calcium after Ang II, indicating that intracellular calcium release was predominantly derived from the thapsygargin-sensitive endoplasmic reticulum. To investigate the channel involved in calcium influx in rat TPMC, we evaluated the effect of VOCC-L blocker verapamil on Ang II-induced elevation in [Ca2⫹]i. Verapamil (Fig. 4C) abolished Ca2⫹ influx determining a low rise of the ion similar to that observed in extracellular Ca2⫹ free experiments. Ang II-induced contraction

Cell areas were analyzed as described in Materials and Methods and percent of contraction was computed as the difference in the areas before and after stimulation. Angiotensin II induced TPMC contraction in a dosedependent fashion with EC50 value of 1.01 ⫻ 10⫺9 m (0.79 ⫺ 1.28 ⫻ 10⫺9 m, 95% confidence limits). Pretreatment with Losartan (100 nm) produced a rightward parallel shift in the dose-response curve for Ang II without affecting the maximal response. Schild analysis of the data gave a pA2 value of 8.156 (8.21 ⫺ 8.10, 95% confidence limits) and a slope of 0.99 (0.86 ⫺1.13, 95% confidence limits), not significantly different from unity (Fig. 5). Ang II-induced contraction was inhibited, with an IC50 of 2 ⫻ 10⫺8 m (Fig. 6), by AM, a vasoactive peptide that we have previously shown to inhibit the ET1-induced contraction in TPMC. The contraction evoked by 100 nm Ang II was slightly less potent than by 100 nm ET1. BQ 123 (100 nm) and Losartan inhibited, respectively, the ET1 and Ang II-induced contraction at similar extent. The protein kinase C (PKC) inhibitor calphostin C (100 nm) markedly blunted the contraction induced by Ang II (Fig. 7). 3

H-thymidine incorporation

Ang II caused a dose-dependent increase in 3H-thymidine incorporation to a maximum, at 1 mm, of 116% above the control value (Fig. 8A). Losartan (100 nm) caused a decrease in 3H-thymidine in-

corporation promoted by 100 nm Ang II, whereas the AT2 antagonist PD123319 did not affect Ang II-induced incorporation (Fig. 9). In the presence of Ang II, the tyrosine kinase inhibitor AG18 was less potent than the MAPK inhibitor PD 98059 in inhibiting 3H-thymidine incorporation into the DNA (Fig. 9A). Cell growth analysis

Stimulation of quiescent testicular peritubular myoid cells with Ang II in virtually serum-free medium for 2 d led to a significant dose-dependent increase in cell number (Fig. 8B). The Ang II-stimulated cell proliferation was completely inhibited by coincubation with Losartan, whereas PD123319 did not affect Ang II-induced proliferation. As in 3H-thymidine incorporation experiments, the tyrosine kinase inhibitor AG18 was less potent than the MAPK inhibitor PD 98059 in inhibiting Ang II-induced proliferation (Fig. 9B). Discussion

To evaluate the possible role of Ang II in the control of testis function, in this paper we investigated its effects on rat peritubular myoid cells (TPMC) in vitro, in terms of contraction and proliferation, and the specific pathways involved. Our study documents that angiotensin II induces contraction and proliferation in rat TPMC. The Ang II effects are mediated by binding to specific receptors. Scatchard analysis showed a single class of binding sites, and competition binding experiments performed with Losartan and PD 123319 indicated that TPMC express only the AT1 receptor type. In other SMCs, Ang II binding to receptors is followed by an increase of intracellular calcium (27). In TPMC, Ang II induced a rise in intracellular Ca2⫹ in a dose-dependent fashion. The rise in intracellular Ca2⫹ was blunted in Ca2⫹free medium and in the presence of voltage-operated calcium channel blockers, indicating that both Ca2⫹ mobilization from intracellular stores, predominantly thapsygarginsensitive endoplasmic reticulum, and extracellular Ca2⫹ influx were involved. Ang II induced TPMC contraction in a dose-dependent

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FIG. 8. A, Ang II induced 3H-thymidine incorporation in testicular peritubular myoid cells. Cells were subcultured and plated in 24well plates at a density of 1 ⫻ 105 cells/well. Cell cycle synchronization was performed in serum-free medium for 24 h before 3 H-thymidine assay. After administration of Ang II for 20 h cells were incubated with 3H-thymidine (1 ␮Ci/ml) for 4 h. The tritium incorporation was determined by scintillation counting. The results are expressed as percent increase in 3H-thymidine incorporation assuming as 100% the incorporation in control cells. Each histogram represents the mean ⫾ SEM of three separate experiments run in quadruplicate. Statistical difference vs. control at P ⬍ 0.001 (***) and P ⬍ 0.01 (**). B, Stimulation of quiescent testicular peritubular myoid cells with Ang II led to a significant dosedependent increase in cell number. Cells were cultured in 24-well plates at a density of 1 ⫻ 105 in virtually serum-free medium for 2 d and stimulated every 24 h with Ang II. Cell number was evaluated in a Thoma’s chamber by counting four wells in triplicate for each treatment and experiments were carried out on three separate cultures. Each histogram represents the mean ⫾ SEM. Statistical difference vs. control at P ⬍ 0.001 (***) and P ⬍ 0.01 (**).

FIG. 9. A, Effect of angiotensin II competitive inhibitors Losartan and PD123319 (100 nM), MAPK kinase (MEK)1/2 inhibitor PD 98095 (5 ␮M) and tyrosine kinase inhibitor AG18 (1 ␮M) on Ang II-induced 3H-thymidine incorporation in testicular peritubular myoid cells. Culture procedure and 3H-thymidine uptake measurement as in the previous figure. Each histogram represents the mean ⫾ SEM of three separate experiments run in triplicate. *, Significant difference at P ⬍ 0.05; and **, significant difference at P ⬍ 0.001 vs. samples treated with angiotensin II only. B, Effect of angiotensin II competitive inhibitors Losartan and PD123319 (100 nM), MEK1/2 inhibitor PD 98095 (5 ␮M) and tyrosine kinase inhibitor AG18 (1 ␮M) on Ang II-induced proliferation in testicular peritubular myoid cells. Cell culture and Ang II addition as in the previous figure. Peptide antagonists, tyrosine kinase inhibitor, or MEKs inhibitor were added to cell culture 20 min before Ang II treatment. Cell number was evaluated in a Thoma’s chamber by counting four wells in triplicate for each treatment and experiments were carried out on three separate cultures. *, Significant difference at P ⬍ 0.05; and **, significant difference at P ⬍ 0.001 vs. samples treated with angiotensin II only.

fashion increasing intracellular calcium and then activating the PKC. Moreover, Ang II-induced contraction was blunted by adrenomedullin, a vasoactive peptide previously shown to be able to induce cAMP production and to inhibit ET1induced contraction in TPMC in vitro (26). As Ang IIpromoted contraction is inhibited by adrenomedullin, the paracrine balance between factors inducing TPMC contraction and those inhibiting it is likely to be complex and to involve several peptides, and, among them, AM may play a prominent role in the regulation of TPMC contraction.

Ang II stimulated TPMC 3H-thymidine incorporation and proliferation in a dose-dependent manner. This was inhibited by Losartan and not by PD 123319. The MAPK family members are ubiquitously expressed protein kinases activated in response to a variety of extracellular stimuli and are implicated in SMC growth as well as differentiation and apoptosis (28). The MAPKs are components of specific kinases cascades characterized by activation by specific stimuli. The MAPKs require dual phosphorylation upon threonine and tyrosine and include


TEY, TP, and TGY sequence. The TEY subgroup includes the ERK1 and ERK2 (29). Activation of ERK1/2 has been considered involved in cell proliferation and differentiation in SMC in response to extracellular stimuli like Ang II (30, 31). In SMC, Ang II induces contraction and growth through receptor-linked pathways that increase intracellular free calcium and pH, and the activation of these second messengers involves tyrosine kinase-dependent signaling pathways (32, 33). To evaluate the contribution of these pathways in TPMC growth, we performed a set of experiments assessing 3Hthymidine incorporation and cell proliferation using the ERK1/2 inhibitor PD 98059 and the tyrosine kinase inhibitor AG18. The involvement of MAP and tyrosine kinases in the Ang II-induced proliferation was shown by the capability of AG18 and PD 98059 to inhibit 3H-thymidine incorporation into DNA and growth in TPMC. The components of the renin angiotensin system have long been known to be expressed in the mammalian testis (34, 35), and their involvement in the development of rat testis has also been documented (36). The AT2 receptor is present, as the major receptor subtype, during the neonatal stage of testicular development and it subsequently shifts to AT1 subtype during puberty (37). TPMC are the main cell type of the seminiferous tubule wall, take part in the paracrine regulation of the testis, contribute at the blood-testicular barrier and, owing to their contractile activity, are responsible for the contraction of seminiferous tubules, responsible, at least partially, for sperm progression in tubular lumen (38). In addition, recently seminiferous tubule contraction has been shown to be dependent upon the seminiferous tubule stage (5, 6). Previous studies have demonstrated that several agonists, such as endothelin-1, arginine vasopressin, TGF␤, platelet-derived growth factor, oxytocin, and prostaglandins, oxytocin, and prostaglandins induce TPMC contraction (4 –7), and, up to now, one peptide, adrenomedullin, has been reported to induce relaxation, in endocrine, paracrine, and autocrine fashion (26). In this context, our investigation documents that Ang II, inducing contraction and TPMC proliferation, and being antagonized by adrenomedullin, may play a role as a new agonist in the paracrine regulation of seminiferous tubule function. In addition, although the pathophysiological role of the Ang II actions on TPMC remains to be elucidated, our data suggest that Ang II-induced TPMC growth might be, to some extent, responsible for the proliferation of those cells in human testicular disorders characterized by thickening of the tubular wall, such as varicocele (39), hypospermatogenesis (40), and cryptorchidism (41). Acknowledgments Received January 18, 2002. Accepted April 16, 2002. Address all correspondence and requests for reprints to: Prof. Vittorio Santiemma, Dipartimento di Fisiopatologia Medica, V Clinica Medica, Policlinico Umberto I, Universita` di Roma “La Sapienza,” Viale del Policlinico, 00161 Roma, Italy. E-mail: [email protected].


This work was partially supported by 60% and 40% Ministero della Ricerca Scientifica e Tecnologica grants.

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