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Biology of Reproduction 64, 619-624 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Adrenomedullin Inhibits the Contraction of Cultured Rat Testicular Peritubular Myoid Cells Induced by Endothelin-11

Vittorio Santiemma2,a, Fabio Rossia, Lara Guerrinia, Athina Markouizoua, Giuseppe Pasimenia, Simonetta Palleschia, and Aldo Fabbrinia

a Dipartimento di Fisiopatologia Medica, Università "La Sapienza" Facoltà di Medicina, 00161 Rome, Italy

ABSTRACT

The present study documents that adrenomedullin (AM), a vasoactive peptide originally identified in pheochromocytoma tissue and present in the testis, in vitro affects the function of testicular peritubular myoid cells (TPMC), a contractile cell type located in the seminiferous tubule wall. AM stimulated cAMP production by cultured TPMC taken from 16-day-old rats, and this effect was completely inhibited by the AM antagonist AM-(22–52) and partially by the CGRP (calcitonin gene-related peptide) antagonist CGRP-(8–37). Studies on TPMC contractile activity documented that AM inhibits TPMC contraction induced by endothelin-1 (ET-1) and that its effect is antagonized by AM-(22–52). Neutralizing AM produced by TPMC with the addition of anti-AM antibody induced a significant increase of ET-1-induced contraction. When exposed to the protein kinase A inhibitor H-89, AM inhibitory activity on ET-1-induced TPMC contraction was suppressed, whereas the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl esther did not modify AM activity. In conclusion, our study indicates that AM stimulates cAMP production and inhibits the contraction induced by ET-1 in TPMC in vitro, and that AM produced by TPMC has an autocrine effect. We propose that AM may have a role in the control of seminiferous tubule contraction.

calcium, cAMP, hormone action, interstitial cells, kinases, signal transduction, sperm motility and transport, testes

INTRODUCTION

Adrenomedullin (AM), a 52-amino acid ringed-structure peptide with C-terminal amidation, is a potent vasodilating agent belonging to the family of calcitonin gene-related peptide (CGRP) and amylin, which was first isolated from human pheochromocytoma tissue [1]. Adrenomedullin is actively secreted by endothelial cells [2], and its production is regulated by many hormones and locally produced substances [3]. Adrenomedullin vasorelaxant effect is mediated either by a direct action on vascular smooth muscle cells (VSMC) through a marked increase of cAMP [4, 5] or by nitric oxide (NO) production by endothelial cells with subsequent VSMC relaxation [6]. However, VSMC, too, secrete AM, although in lesser amounts than endothelial cells [7].

In the rat, the AM gene is expressed in several tissues such as lung, adrenal medulla, heart, kidney, spleen, and ovary [8, 9], and AM immunoreactivity is present in the lung, heart, adrenal medulla, brain, submandibular glands, aorta, kidney, spleen, testis, duodenum, pancreas, and liver [10]. Prominent AM effects, beyond vasorelaxation, identified so far include promotion of diuresis and natriuresis [11] and inhibition of bronchoconstriction [12]. The low AM plasma concentration (~2 fmol/ml in the rat) suggests that the AM effects in the different tissues are likely to be due to local production.

Recently, the presence of AM receptors has been documented in testicular peritubular myoid cells (TPMC) [13]. Testicular peritubular myoid cells are the main cellular component of the seminiferous tubule wall and take active part in the paracrine regulation of testis function. They cooperate with Sertoli cells in the production of the basal membrane of the tubule and deeply affect Sertoli cell function by secreting paracrine factors that enhance Sertoli cell secretory activity [14]. In addition, TPMC are contractile cells that express the cytoskeletal markers of true smooth muscle cells (SMC), such as {alpha}-isoactin, F-actin, and myosin [15, 16]. Their contractile activity is responsible for the contraction of seminiferous tubules and, at least partially, for sperm release during spermiation. Contraction of TPMC is regulated in an endocrine, paracrine and autocrine fashion by several factors [17]. Among them, endothelin-1 (ET-1) has been documented to induce a Ca2+ transient in TPMC and to stimulate TPMC contraction in a dose-dependent manner via a protein kinase C pathway [18].

The aim of the present study was to investigate whether TPMC are a target for AM, and whether AM is capable to affect TPMC contraction induced by ET-1.

MATERIALS AND METHODS

Materials

The rAM, AM-(22–52), rCGRP, CGRP-(8–37), and polyclonal AM antibody were purchased from Peptide Institute (Osaka, Japan). N-[2-(p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was purchased from Calbiochem (Milan, Italy) and NG-nitro-L-arginine methyl esther (L-NAME) from Sigma (Milan, Italy).

Rat TPMC Isolation and Culture

Rat TPMC were isolated from 16-day-old Sprague-Dawley rats provided by Charles River (Calco, Italy). The rats were killed by cervical dislocation immediately after delivery. Testes were aseptically removed, decapsulated, and finely minced. After sequential enzymatic digestion with 0.25% trypsin and 0.1% collagenase (Boehringer Mannheim, Germany) [19], purified peritubular myoid cells were obtained by a Percoll (Pharmacia, Uppsala, Sweden) discontinuous gradient technique as previously described [15]. Briefly, the cells derived from the collagenase supernatant were layered on the top of the Percoll gradient (30 to 70%). After centrifugation at 3000 rpm for 30 min, the third band separated containing purified rat TPMC was aspirated and the cells were washed and plated. After cell separation, rat TPMC viability was assessed by the trypan blue exclusion test. To evaluate cell purity [20], alkaline phosphatase activity was tested on adherent cells and was never below 95%. The contamination by endothelial cells, evaluated by immunocytochemistry with a rabbit anti-human von Willebrand factor antibody (Sigma), was 1% at Day 1 of culture, and after 3 days positive cells were no longer present. Primary cultures of rat TPMC were maintained at 32°C in 5% CO2 atmosphere in Dulbeccos modified essential medium (DMEM)/F12 (1:1) containing 10% fetal calf serum (Gibco, Grand Island, NY), and the medium was changed every 48 h.

Determination of cAMP

Confluent TPMC (2 x 105 cells/well in 24-well trays) were washed twice with the assay buffer (DMEM containing 0.1 % BSA, 20 mM Hepes, and 0.5 mM isobutyl-methylxanthine, pH 7.4) and subsequently incubated for 15 min at 37°C in the absence or in the presence of AM, CGRP, and antagonists as reported in the Results. Cells were lysed in 6% trichloroacetic acid, and the acid was removed by water-saturated diethyl ether extraction. After lyophilization of the aqueous phase, intracellular cAMP was measured by an RIA kit (Amersham, England).

Collagen Gel Contraction

Cell contraction was evaluated after 6–8 days of culture according to a previously described method [21]. The TPMC were detached with trypsin 0.025% (Sigma), suspended at a concentration of 2 x 106/ml of Hanks, and mixed to a solution consisting of 10-strength MEM, 0.26 M NaHCO3, and 0.2 % type I collagen. A final cell density of 4 x 105 was obtained by the addition of sterile water. Aliquots (0.5 ml) of this cell suspension were pipetted into 24-multiwell tissue culture plates previously coated with 0.67% agarose. After the mixture gelled (37°C), MEM containing AM or CGRP (for the concentrations in each experiment, see Results) and, when appropriate, the antagonists AM-(22–52) and CGRP-(8–37) were layered onto the gels. In a set of experiments the polyclonal AM antibody was added before gelling. Endothelin-1 was added after 1 h, and gel contraction was evaluated after 3 h of incubation (time of the maximal response). For measurements, the plates were videocaptured (Screen Machine II; FAST Multimedia AG, Munchen, Germany), the areas of the collagen gels were calculated from the digitized images (SigmaScan Pro; Jandel Scientific, Erkrath, Germany), and contraction (C) computed according to the formula:

where a is the area of the collagen gel and A is the well's area.

The viability of TPMC in the gels was evaluated digesting the collagen matrix by adding type VII Clostridium histolyticum collagenase to each well and incubating at 37°C for 1 h. Cells obtained from the gels were assessed by the trypan blue exclusion test and counted.

Microperfusion Chamber System

A glass coverslip with plated TPMC 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 a Nikon microscope using a 20x phase-contrast objective. Images were videocaptured by a Panasonic B/W camera coupled to a Saba video recorder. After perfusion (1 ml/min) with regular buffer for 3 min, time necessary for thermal equilibration, cells were flushed with test compounds.

Statistical Analysis

Results are presented as mean ± SEM. Student's unpaired t-test was used to assess the significance of the differences between means, and P < 0.05 was taken as statistically significant.

RESULTS

Cyclic AMP Production

Adrenomedullin induced cAMP production in a dose-dependent fashion (Fig. 1) with a sixfold maximal increase over control levels. Adrenomedullin was more potent (~60%) than CGRP in stimulating cAMP production. The 50% effective concentration (EC50) value was 8 ± 1.3 nM for AM and 33.5 ± 1.7 nM for CGRP. In addition, the cAMP response evoked by AM was completely inhibited by the antagonist AM-(22–52) with a 50% inhibitory concentration (IC50) of 29 ± 1.6 nM, and the CGRP antagonist CGRP-(8–37) was only partially (~50%) able to inhibit the AM-induced cAMP production (Fig. 2), but completely abolished the response to CGRP (data not shown).



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  		FIG. 1. Stimulation of cAMP production by rAM and rCGRP in rat TPMC. Confluent TPMC were incubated for 15 min at 37°C in the assay buffer containing 0.5 mM isobutyl-methylxanthine in the absence or in the presence of different concentrations of rAM (circles) and rCGRP (squares). Each point is expressed as the mean of two separate experiments run in triplicate; bars show SEM



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  		FIG. 2. Effect of AM and CGRP antagonists on cAMP production stimulated by rAM in cultured rat TPMC. Confluent TPMC were incubated in the absence or in the presence of AM-(22–52) (circles) and CGRP-(8–37) (squares) at indicated concentrations. Each point is expressed as the mean of two separate experiments run in triplicate; bars show SEM

Effect of AM on TPMC Contraction

To investigate the effect of AM on TPMC contraction, ET-1 was chosen as the contractile agent, as its activity on cultured TPMC has already been documented [18]. A preliminary study performed in a microperfusion chamber system showed that the marked contraction of TPMC induced by ET-1 was effectively inhibited by AM (Fig. 3). Subsequently, a study based on collagen gel contraction was performed to measure the AM effect seen with the microperfusion system. Adrenomedullin was able to inhibit the gel contraction induced by 10 nM ET-1 in a dose-related manner. The effect was evident at 1 nM and maximal at 100 nM (Fig. 4). These results were supported by the effect of AM on TPMC contraction induced by increasing concentrations of ET-1. The addition of 10 nM AM completely inhibited the response to 1 nM ET-1 and lower concentrations (Fig. 5). Adrenomedullin-(22–52) antagonized the AM effect on ET-1-induced TPMC contraction in a dose-dependent manner with an IC50 of 23 ± 0.5 nM (Fig. 6).



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  		FIG. 3. Inhibitory effect of AM on TPMC contraction induced by ET-1. Endothelin-1 added to the perfusate provoked a marked cell contraction leading to the detachment of many cells from the coverslip (a–c). In AM-pretreated cells, ET-1 induced a slight contraction of a few cells only (d–f). Magnification x240.



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  		FIG. 4. Dose-dependent effect of AM on rat TPMC contraction induced by ET-1. Aliquots of 2 x 105 cells/well in 0.5 ml of a 1.6-mg/ml type I collagen solution were exposed to rAM at indicated concentrations. Endothelin-1 (10 nM) was added after 1 h and gel contraction was measured after 3 h. Each point is expressed as the mean of two separate experiments run in quadruplicate; bars show SEM



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  		FIG. 5. Effect of rAM on TPMC contraction induced by increasing concentrations of ET-1 (squares). An aliquot of 10 nM AM (circles) was layered onto the gels, and after 1 h, ET-1 was added at indicated concentrations. Each point is expressed as the mean of two separate experiments run in quadruplicate; bars show SEM



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  		FIG. 6. Dose-related effect of AM-(22–52) on AM inhibition of rat TPMC contraction induced by ET-1. Aliquots of 10 nM AM and increasing concentrations of rAM-(22–52) were layered onto the gels. After 1 h, 10 nM ET-1 was added. Each point is expressed as the mean of two separate experiments run in quadruplicate; bars show SEM

To evaluate whether AM produced by TPMC might interfere with ET-1-induced contraction, experiments were run adding anti-AM antibody to the final collagen solution at 1:50 and 1:100 dilutions. The antibody addition increased the gel contraction induced by 100 nM ET-1 (Fig. 7).



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  		FIG. 7. Effect of TPMC exposure to rat AM antibody on the contraction induced by 100 nM ET-1. Each point is expressed as the mean of two separate experiments run in quadruplicate; bars show SEM. **P <= 0.01 versus C (controls)

Finally, we evaluated the effect of the protein kinase A inhibitor H-89 and of the NO synthase inhibitor L-NAME on TPMC contraction in order to investigate the signal transduction pathway of the AM action. H-89 inhibited the AM effect, whereas L-NAME was ineffective (Fig. 8).



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  		FIG. 8. Effect of PKA and NO synthase inhibition on AM blunting of TPMC contraction induced by ET-1. H-89 (10 µM) or L-NAME (0.1 mM) was layered onto the gels. Aliquots of 10 nM AM and 10 nM ET-1 were added after 30 min and 1 h, respectively. Each point is expressed as the mean of two separate experiments run in quadruplicate; bars show SEM. **P <= 0.01 versus C (controls)

DISCUSSION

Our study documents that in TPMC in vitro AM elicits cAMP accumulation and antagonizes the contraction induced by ET-1. In addition, it indicates that TPMC secrete AM that has an autocrine effect.

Adrenomedullin promoted cAMP production in TPMC, and its effect was inhibited by the AM antagonist AM-(22–52) in a dose-related fashion up to complete suppression, and only partially antagonized (less than 50%) by CGRP-(8–37). Altogether, these results suggest that AM is likely to act on TPMC through both AM and CGRP1 receptors. However, the weaker action of CGRP as compared to that of AM suggests the prevalence of AM receptors. In TPMC, AM action seems not to be mediated by cytosolic Ca2+, as AM did not change baseline [Ca2+]i nor the Ca2+ transient induced by ET-1 (data not shown).

Adrenomedullin has been reported to increase cAMP in several SMC types, including VSMC [4, 5, 22–24], mesangial cells [25–27], myometrial cells [28], and cecal circular SMC [29]. More controversial is the effect of AM on [Ca2+]i in SMC. A decrease of [Ca2+]i induced by AM has been documented in rings of pig coronary arterial smooth muscle [30] but not in cultured VSMC [4, 5]. In bovine and rat endothelial cells, by contrast, AM increases [Ca2+]i, stimulating Ca2+ mobilization from intracellular thapsigargin-sensitive storage and influx through the ion channels [6, 23].

The present study documents that AM strongly inhibits TPMC contraction induced by ET-1 and that its effect is inhibited by the AM antagonist AM-(22–52). To the best of our knowledge, this is the first report of an inhibitory effect of AM on the contraction of SMC elicited by ET-1. Experiments are ongoing in our laboratory to investigate whether AM is able to antagonize TPMC contraction provoked by other agonists.

Previous studies have demonstrated that AM has a potent hypotensive and vasorelaxant effect in vivo [1, 31–33] and in vitro [30, 34]. The vasorelaxant effect has been shown to depend on the presence of the endothelium, as in rat aorta, or to be direct on VSMC, as in porcine coronary artery [6]. In the former instance, AM action is thought to be mediated by NO, in the latter by the increase of cAMP production.

We therefore investigated whether the AM effect on TPMC is mediated by cAMP or NO. Testicular peritubular myoid cells were exposed to PKA inhibitor H-89, which was able to suppress the AM inhibitory effect on ET-1-induced TPMC contraction, whereas the NO synthase inhibitor L-NAME did not affect AM action on TPMC.

This is consistent with the observation that the activation of receptors associated with G proteins coupled to adenlylcyclase, subsequent generation of cAMP, and activation of cAMP-dependent protein kinases results in inhibition of smooth muscle contraction [35]. Phosphorylation of smooth muscle myosin light chain phosphatase resulting in inactivation is a mechanism proposed [36].

Testicular peritubular myoid cell contraction has long been held responsible for the contraction of the seminiferous tubules [37]. Recently, it has been shown that seminiferous tubule contraction may be dependent upon the seminiferous tubule stage. In the case of ET-1-induced contraction [38], ET-1 production is stage dependent, whereas in the case of oxytocin its effect is stage dependent [39].

We previously documented that ET-1, secreted by Sertoli cells [40], induces the contraction of TPMC [18], and proposed that it is relevant in the paracrine regulation of testis function.

Our data indicate that TPMC are likely to be a source of the AM present in the testis, and AM receptors have already been reported in TPMC. Currently, it is not known whether other testicular cell types secrete AM. Thus, in the first instance, it is legitimate to speculate that AM plays an autocrine role in the testis. In this regard it is very interesting that AM is able to antagonize TPMC contraction induced by ET-1, as up to now only agents inducing the contraction of TPMC have been reported [17], whereas many smooth cell types are known to be regulated by a balance of factors inducing contraction and those inhibiting it. The capability of AM to modulate the TPMC contractile response to ET-1 may be an important factor in the regulation of seminiferous tubule contraction in different stages of spermatogenesis. In addition, further investigation is needed to assess whether AM has a paracrine effect, e.g., on Sertoli cell production of ET-1, beyond the autocrine role presently reported.

Caution is needed to extend to adult TPMC the present findings, as our cells came from animals just engaged in the spermatogenic process (Sertoli cells cease to replicate at Day 15 after birth [41]). However, TPMC microfilaments, examined by electron microscopy, reach the adult aspect around Day 15 [42], and other markers, such as desmin, alkaline phosphatase activity, and {alpha}-isoactin, a marker of terminal differentiation in SMC, are already present at that age [15,16].

In conclusion, the finding that AM is able to antagonize the TPMC contraction induced by ET-1 suggests that AM may be part of the local control of the seminiferous tubule, and opens the way to further investigation in this field.

FOOTNOTES

First decision: 14 October 1999.

1 This work was partially supported by 60% and 40% MURST grants. Back

2 Correspondence: Vittorio Santiemma, Dipartimento di Fisiopatologia Medica, V Clinica Medica, Policlinico Umberto I, Universita’ "La Sapienza," Viale del Policlinico, 00161 Roma, Italy. FAX: 39 06 490530; v.santiemma{at}caspur.it Back

Accepted: September 22, 2000.

Received: July 29, 1999.

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