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Comparison of endothelin-1-mediated tissue tension and calcium mobilization effects in isolated rabbit corpus cavernosum
BASIC SCIENCE
COMPARISON OF ENDOTHELIN-1-MEDIATED TISSUE TENSION AND CALCIUM MOBILIZATION EFFECTS IN ISOLATED RABBIT CORPUS CAVERNOSUM HARINDRA R. ABEYSINGHE, JOANNA CLANCY,
AND
YUHONG QIU
ABSTRACT Objectives. To directly compare and contrast the effects of endothelin-1 (ET-1) and adrenoreceptor agonists norepinephrine and phenylephrine on eliciting calcium influx in primary rabbit corpus cavernosum cells and their ability to elicit tissue contractions. The potent vasoconstrictor peptide ET-1 and the alphaadrenoreceptor agonists are important modulators of smooth muscle tone in the penile corpus cavernosum. However, the mechanisms involved in maintaining smooth muscle tone and contraction are not clearly understood. Methods. Intracellular calcium mobilization was measured in cultured corpus cavernosum smooth muscle cells using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader. Tissue tension studies on rabbit corpus cavernosum were conducted using organ chambers. Results. ET-1 at concentrations as low as 10 nM was sufficient to induce a transient increase of intracellular calcium in these cells. In contrast, concentrations of 1 mM and greater of norepinephrine and phenylephrine were required to elicit comparable calcium fluxes in cavernosum cells. Tissue bath studies indicated that ET-1 is a potent stimulator of corpus cavernosum smooth muscle contraction, with concentrations as low as 10 nM sufficient to initiate contraction. Conclusions. The potency of ET-1 in producing contraction on tissue strips and inducing calcium flux suggests that ET-1 might be an important mediator for modulating and maintaining corpus cavernosum smooth muscle tone. Therefore, additional exploration of the role of endothelins and their receptors in the tumescence and detumescence states of the penis would be extremely valuable. UROLOGY 60: 925–930, 2002. © 2002, Elsevier Science Inc.
N
ormal penile erection, a hemodynamic event involving both the central nervous system and local factors, is regulated by relaxation of the cavernosal arteries and corporal smooth muscle. Trabecular smooth muscle tone is tightly regulated by cholinergic, adrenergic, and nonadrenergicnoncholinergic (NANC) neurotransmitters.1–7 Studies have shown that after penile erection, the process of detumescence is regulated, at least in part, by the release of norepinephrine (NE) from the sympathetic nerves, resulting in activation of the alpha-adrenoreceptors in cavernosum tissue.8,9 From the Department of Reproductive Therapeutics, Johnson & Johnson Pharmaceutical Research & Development, Raritan, New Jersey Reprint requests: Harindra R. Abeysinghe, Ph.D., Johnson & Johnson Pharmaceutical Research & Development, Route 202, P.O. Box 300, Raritan, NJ 08869 Submitted: September 20, 2001, accepted (with revisions): May 16, 2002 © 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
However, it is not clear whether these nerves and signaling pathways are also responsible for providing constant tone to the cavernosal smooth muscle involved in maintaining penile flaccidity. Recently, attention has focused on the role of endothelin-1 (ET-1) as a potential modulator of corpus cavernosum basal tone and its importance in maintaining smooth muscle cell contractility when the penis is in the flaccid state. Studies have implicated ET-1 as a potent agonist for vascular smooth muscle cell contraction.10 –13 ET-1 is a 21-amino acid peptide secreted from the endothelial cells that line the walls of vascular tissue.14 –16 Similar to other vasoconstrictors, the physiologic effects of ET-1 are mediated by increasing intracellular calcium concentrations in cells. It is believed that, when released from endothelial cells into the surrounding tissue, ET-1 binds its receptor at the target cells and facilitates an increase in calcium by the release from intracel0090-4295/02/$22.00 PII S0090-4295(02)01845-9 925
lular calcium stores using the inositol triphosphate pathway and by direct influx using voltage-gated calcium channels.17–19 In human corpus cavernosum smooth muscle, ET-1 has been shown to elevate cytosolic and nuclear calcium levels in cells.10,20,21 The elevation of nuclear calcium is believed to play a role in inducing the expression of some of the immediate early genes such as c-myc and c-fos in human corpus cavernosum smooth muscle cells.21 It has also been demonstrated that ET-1 can increase cell proliferation in culture, which could be abrogated by blocking ET-1 receptors, as well as calcium channels.21 These observations suggest that ET-1 may be involved in regulating multiple physiologic pathways that are intimately linked to intracellular calcium regulation. In separately published reports, Christ et al.10 and Zhao and Christ20 have shown ET-1 to be a potent agonist for smooth muscle contraction in tissue, as well as for calcium flux in cells from corpus cavernosum. However, no direct comparison has been made for the effect of ET-1 on these two physiologic parameters. The purpose of this study was to investigate further the role of ET-1 and its receptor activity in cavernosal tissue and cells by measuring the physiologic responses to ET-1. For tissue studies, rabbit corporal tissue was excised, and tissue contraction in response to ET-1 and other agonists was measured in organ chambers. For cellular studies, primary myocyte cultures were established from rabbit corporal tissue, and changes in intracellular calcium levels induced by ET-1 were measured using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader (FLIPR). MATERIAL AND METHODS Corpus cavernosum tissue was obtained from sexually mature male New Zealand white rabbits. The animals were killed with a lethal injection of pentobarbital, and radical penectomy was performed. Rabbit corpus cavernosum smooth muscle cells were cultured following the procedures of Moreland et al.,22 with minor modifications.23 In brief, corporal tissue sections were washed several times in Hank’s balanced salt solution and then placed into a 10-cm tissue culture dish containing Dulbecco’s modified Eagles medium. The tissue pieces were mechanically separated and minced using a sharp scalpel. Approximately 1-mm3 pieces were transferred to six-well cluster plates, and cells growing out from the explants were cultured.
IMMUNOCYTOCHEMISTRY FOR SMOOTH MUSCLE CELL PROTEINS Cells were grown on coverslips, fixed, permeabilized, and stained with anti-desmin II mouse monoclonal antibody (ICN Pharmaceuticals, Aurora, Ohio) and anti-alpha-actin, smooth muscle mouse monoclonal antibodies (Calbiochem, San Diego, Calif), and visualized by fluorescence microscopy. 926
MEASUREMENT OF INTRACELLULAR CALCIUM FLUX USING FLIPR Intracellular calcium flux was measured using a FLIPR 384 following the manufacturer’s instructions (Molecular Devices, Sunnyvale, Calif). Corpus cavernosum cells were seeded into black-walled clear-base 96-well plates at a density of 20,000 cells per well. The next day, the medium was removed, and the cells were incubated for 1 hour at 37°C in the calcium chelating dye fluo-3AM (Molecular Probes, Eugene, Ore) mixed with a buffer solution. The dye solution was removed, and the cells were washed twice with buffer solution. ET-1, NE, and phenylephrine (PE, Sigma-Aldrich, St. Louis, Mo) were mixed with the buffer solution at the desired concentrations and separated into aliquots in triplicate into a 96-well compound plate. The 96-well plates were loaded into the FLIPR, and fluorescence readings were obtained at timed intervals. Liquid additions were made with an automated 96-channel pipetter in the FLIPR.
RABBIT CORPUS CAVERNOSUM TISSUE TENSION STUDIES Rabbit corpus cavernosum tissue was carefully dissected free from the surrounding tunica albuginea and cut into strips of approximately 0.3 ⫻ 0.3 ⫻ 1.2 cm3 and bathed in Krebs solution. The tissue strips were vertically mounted in organ baths with one end attached to an isometric transducer and the other to a glass hook at the bottom of the bath. The tissue strips were immersed in warmed (37°C) and oxygenated (95% oxygen plus 5% carbon dioxide) Krebs solution. Varying concentrations of ET-1 and PE were added directly to the organ baths, and the changes in isometric tension were recorded on a computer. The tissue tension measurements for each respective treatment of ET-1 and PE were performed on separate tissue strips, and the mean contraction for each treatment is reported from three separate contractions.
RESULTS Rabbit corpus cavernosum cells were characterized by immunofluorescence staining for muscle cell-specific filament proteins, desmin and alphaactin. The cells displayed intense longitudinal staining for both filament proteins (Fig. 1). It was estimated that between 90% and 95% of cells stained positive for both muscle cell markers. The addition of ET-1 to corpus cavernosum smooth muscle cells resulted in a transient increase of intracellular calcium (Fig. 2). The increases in calcium were dose dependent for ET-1, with a 100-nM concentration producing the maximal increase in fluorescence intensity. Treatment concentrations greater than 100 nM did not show a significant increase above the peak fluorescence for 100-nM ET-1 (data not shown). The lowest concentration of ET-1 capable of eliciting a calcium response was 5 nM. ET-1-induced calcium responses were completely abolished when the cells were treated with the ET-1 receptor antagonist PD145065 (data not shown). The conventional adrenoreceptor agonists NE and its synthetic analog PE were applied at different concentrations to the rabbit corpus cavernosum smooth muscle cells to determine the relative changes of intracellular calcium (individual fluoUROLOGY 60 (5), 2002
A
B
FIGURE 1. Immunocytochemistry on rabbit corpus cavernosum smooth muscle cell cultures. Cells were seeded onto coverslips and incubated with monoclonal antibodies recognizing (A) mouse desmin II and (B) smooth muscle alpha-actin followed by staining with fluorescein-conjugated antibodies to mouse IgG. Positive staining for actin and desmin intermediate filament proteins are clearly visible in cells.
FIGURE 2. ET-1 induction of a dose-dependent calcium response in corpus cavernosum cells. Results show fluorescence measurements taken from a single 96-well plate in which ET-1 was added to the cells at the indicated concentrations after 10 seconds. For the control, a similar volume of vehicle used to dissolve ET-1 was added to the cells. The data for each response curve are from a single representative experiment that was repeated three times.
rescence graphs not shown). A comparison of the calcium responses elicited by the different agonists ET-1, NE, and PE in the rabbit corpus cavernosum cells are profiled in Figure 3. The peak fluorescence unit recorded in response to each of the respective agonists is reported as a percentage of the UROLOGY 60 (5), 2002
maximal ET-1 response at the 100-nM dose. Treatment of cells with NE showed a dose-responsive calcium increase, with the maximal increases in cellular calcium occurring at 6 mM. The minimal concentration of NE required to elicit a measurable calcium response was 0.5 mM. For PE treatments 927
FIGURE 3. Comparison of the dose-response changes for intracellular calcium induced by the different agonists. Columns represent the percentage of fluorescence relative to a control treatment of 100 nM ET-1. ET-1 was clearly the most potent of the three agonists with 1 ⫻ 105 and 1 ⫻ 106 greater potency (at direct molar comparisons) than NE or PE, respectively.
at a similar concentration range, the calcium responses were significantly decreased. ET-1 produced concentration-dependent contractions in corpus cavernosum tissue when added to the organ baths (Fig. 4). Concentrations as low as 10 nM ET-1 were sufficient to increase tension more than twofold over baseline. A maximal contraction of almost eightfold over baseline was achieved at the 100-nM ET-1 dose. A 10-M dose of PE was required to elicit a muscle contraction of similar magnitude. PE at 1 M produced a contraction that was almost one half that, and a 100-nM dose did not produce any contraction of the tissue. The maximal contraction was achieved at the 10-M dose of PE; concentrations greater than this did not increase the amplitude of the contraction (data not shown). COMMENT The results of the present study demonstrate that ET-1 is a potent agonist for smooth muscle contraction in rabbit corpus cavernosum. The focus of this study was to examine some of the physiologic effects of ET-1 on corpus cavernosum cells and tissue. Interest has been growing in the potential role of ET-1 in modulating smooth muscle tone 928
when the penis is in the flaccid state. It has been widely speculated that a nonadrenergic mechanism may be involved in maintaining basal tone when the penis is in a flaccid state.10 –13 The results of our study demonstrate that transient increases of intracellular calcium are potentiated by ET-1 at concentrations as low as 10 nM. Increased calcium flux was dose dependent, with maximal increases seen at the 100-nM concentration level. To achieve a similar level of calcium flux in these rabbit corpus cavernosum cells, the adrenoreceptor agonists such as noradrenaline (NE) and its synthetic analog PE had to be applied at concentrations of 1 mM and greater. This observation is in accordance with previous reports indicating that ET-1 is considerably more potent than NE on a molar basis.6,13 However, our results clearly demonstrate that ET-1 is approximately 100,000 times more potent than NE and 1,000,000 times more potent than PE (by direct comparison of molar concentrations) in its ability to elicit a robust calcium response in the cavernosum cells. We discovered that to elicit a robust calcium response in the corpus cavernosum cells, the adrenoreceptor agonists had to be applied at very high concentrations, well above physiologic levels. A UROLOGY 60 (5), 2002
FIGURE 4. Changes in tissue tension for rabbit corpus cavernosum in response to contractile agonists and antagonists. Contraction and relaxation of tissue is presented in grams of tension. Basal tension for each of the tissues was adjusted such that all tissues began at approximately 1 g. Dose-response contraction was observed when tissues were treated with different concentrations of ET-1. Similar levels of contraction were achieved with 10 M PE and 100 nM ET-1.
possible explanation for the dramatic difference in calcium responses between NE and ET-1 in these cells is that ET-1 may be a primary mediator for modulating smooth muscle cell contraction and maintaining basal tone. This is supported by the data derived from the tissue tension studies and the calcium flux studies. The corpus cavernosum tissue responds with a high degree of sensitivity to PE at a concentration of 10 M, resulting in robust contractile activity. However, when cultured cells are treated with PE at this concentration range, no changes in intracellular calcium were detectable. It has been well documented that smooth muscle contraction, like all muscle, requires an increase of intracellular calcium to propagate a contraction.24 As shown in this study, to elicit a significant and detectable calcium flux in the cavernosum cells, the concentration levels of the adrenoreceptor agonists had to be augmented. However, as mentioned previously, the concentration of PE required to elicit a significant corporal smooth muscle contraction in muscle strips was 10 M, almost 100fold less. Intact corporal tissue strips, unlike cells in culture, consist of a heterogeneous mixture of corporal smooth muscle cells, vascular smooth UROLOGY 60 (5), 2002
muscle cells, endothelial cells, and fibroblasts. All these cell types together form an integral component of the cavernosum and have important structural and functional roles in this tissue. It is possible that the primary or immediate effect of PE is not directly on the corpus cavernosum smooth muscle cells, but instead, signals the release of other factors such as ET-1 from the endothelial cells. The local release of ET-1 then initiates the contraction of the cavernosal smooth muscle cells, thus maintaining the penis in a flaccid state. It is also plausible that PE and ET-1 may have a synergistic effect for the induction of smooth muscle contraction. This observation has been previously reported by Christ et al.10 from tissue tension studies on corporal tissue sections obtained from patients undergoing penile surgery. Their study revealed that coadministration of PE and ET-1 produced much greater contractile responses than those observed for PE alone. Regardless of whether ET-1 acts synergistically with other ligands or by itself, the results presented here clearly indicate that it is extremely potent in its ability to generate a calcium flux in cavernosum cells contraction. On the basis of published reports and our findings, it 929
seems plausible that ET-1 and ET-1 receptors may play an important physiologic role in corpus cavernosum smooth muscle function. CONCLUSIONS In this study, we found that ET-1 has a profound effect on corporal smooth muscle cell physiology in terms of its ability to both mobilize calcium and contract tissue rapidly. Its high potency compared with the adrenoreceptor agonists suggests that it may be an important mediator for maintaining smooth muscle cell basal tone in the penis. Additional studies to evaluate the in vitro and in vivo effects of ET-1 and their receptors is required to aid in the understanding of their precise physiologic role in corpus cavernosum smooth muscle contraction. These studies will also be beneficial in determining whether manipulation of ET-1 and its receptors will be useful for therapeutic purposes for male erectile dysfunction. ACKNOWLEDGMENT. To Dr. Joseph Gunnet for his assistance with the calcium imaging studies and Muh-Tsann Lai for technical assistance. REFERENCES 1. Adaikan PG, and Karim SMM: Adrenoreceptors in the human penis. J Auton Pharmacol 1: 199 –203, 1981. 2. Christ GJ, Maayani S, Valcic M, et al: Pharmacological studies of human erectile tissue: characteristics of spontaneous contractions and alterations in alpha-adrenoreceptor responsiveness with age and disease in isolated tissues. Br J Pharmacol 101: 375–381, 1990. 3. Diederichs W, Steif CG, Lue TF, et al: Norepinephrine involvement in penile detumescence. J Urol 143: 1264 –1266, 1990. 4. Hedlund H, and Andersson KE: Comparison of the responses to drugs acting on adrenoreceptors and muscarinic receptors in human isolated corpus cavernosum and cavernous artery. J Auton Pharmacol 5: 81–88, 1985. 5. Hedlund H, Andersson KE, and Mattiasson A: Pre- and post-junctional adreno- and muscarinic receptor function in isolated corpus spongiosum urethrae. J Auton Pharmacol 4: 241–246, 1984. 6. Saenz de Tejada I, Kim N, Lagan I, et al: Regulation of adrenergic activity in penile corpus cavernosum. J Urol 142: 1117–1121, 1989. 7. Saenz de Tejada I, Blanco R, Goldstein I, et al: Cholinergic neurotransmission in human corpus cavernosum. I. Responses of isolated tissue. Am J Physiol 254: 459 –467, 1988. 8. Krane RJ, Goldstein I, and Saenz de Tejada I: Impotence. N Engl J Med 321: 1648 –1659, 1989.
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9. Lue TF, and Tanagho EA: Physiology of erection and pharmacological management of impotence. J Urol 137: 829 – 836, 1987. 10. Christ GJ, Lerner SE, Kim DC, et al: Endothelin-1 as a putative modulator of erectile dysfunction: characteristics of contraction of isolated corporal tissue strips. J Urol 153: 1998 –2003, 1995. 11. Saenz de Tejada I, Carson MP, de las Morenas A, et al: Endothelin: localization, synthesis, activity, and receptor types in human penile corpus cavernosum. Am J Physiol 261: 1078 –1085, 1991. 12. Lau LC, Adaikan PG, and Ratnam SS: Effect of endothelin-1 on the human corpus cavernosum and penile vasculature. Asia Pac J Pharmacol 6: 287–292, 1991. 13. Holmquist F, Andersson K-E, and Hedlund H: Actions of endothelin on isolated corpus cavernosum from rabbit and man. Acta Physiol Scand 139: 113–122, 1990. 14. Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (Lond) 332: 411–415, 1988. 15. Masaki T: The discovery, the present state and the future prospects of endothelin. J Cardiovasc Pharamacol Suppl 13: 1–4, 1989. 16. Inoue A, Yanagisawa M, Kimura S, et al: The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 86: 2863–2867, 1989. 17. Marsden PA, Denthuluri NR, Brenner BM, et al: Endothelin action on vascular smooth muscle involves inositol triphosphate and calcium mobilization. Biochem Biophys Res Commun 158: 86 –93, 1989. 18. Berridge MJ: Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56: 159 –193, 1987. 19. Rasmussen H, Takuwa Y, and Park S: Protein kinase C in the regulation of smooth muscle contraction. FASEB J 1: 177–185, 1987. 20. Zhao W, and Christ GJ: Endothelin-1 as a putative modulator of erectile dysfunction. II. Calcium mobilization in cultured human corporal smooth muscle cells. J Urol 154: 1571–1579, 1995. 21. Giraldi A, Serels S, Autieri M, et al: Endothelin-1 as a putative modulator of gene expression and cellular physiology in cultured human corporal smooth muscle cells. J Urol 160: 1856 –1862, 1998. 22. Moreland RB, Traish AM, McMillin MA, et al: PGE suppresses the induction of collagen synthesis by transforming growth factor 1 in human corpus cavernosum smooth muscle. J Urol 153: 826 –834, 1995. 23. Traish AM, Moreland RB, Gallant C, et al: G-proteincoupled receptor agonists augment adenylyl cyclase activity induced by forskolin in human corpus cavernosum smooth muscle cells. Recep Signal Trans 7: 121–132, 1997. 24. Daniel E, Grover A, and Kwan C: Calcium, in Stephens NL (Ed): Biochemistry of Smooth Muscle. Boca Raton, Florida, CRC Press, 1983, vol III, pp 1–88.
UROLOGY 60 (5), 2002
COMPARISON OF ENDOTHELIN-1-MEDIATED TISSUE TENSION AND CALCIUM MOBILIZATION EFFECTS IN ISOLATED RABBIT CORPUS CAVERNOSUM HARINDRA R. ABEYSINGHE, JOANNA CLANCY,
AND
YUHONG QIU
ABSTRACT Objectives. To directly compare and contrast the effects of endothelin-1 (ET-1) and adrenoreceptor agonists norepinephrine and phenylephrine on eliciting calcium influx in primary rabbit corpus cavernosum cells and their ability to elicit tissue contractions. The potent vasoconstrictor peptide ET-1 and the alphaadrenoreceptor agonists are important modulators of smooth muscle tone in the penile corpus cavernosum. However, the mechanisms involved in maintaining smooth muscle tone and contraction are not clearly understood. Methods. Intracellular calcium mobilization was measured in cultured corpus cavernosum smooth muscle cells using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader. Tissue tension studies on rabbit corpus cavernosum were conducted using organ chambers. Results. ET-1 at concentrations as low as 10 nM was sufficient to induce a transient increase of intracellular calcium in these cells. In contrast, concentrations of 1 mM and greater of norepinephrine and phenylephrine were required to elicit comparable calcium fluxes in cavernosum cells. Tissue bath studies indicated that ET-1 is a potent stimulator of corpus cavernosum smooth muscle contraction, with concentrations as low as 10 nM sufficient to initiate contraction. Conclusions. The potency of ET-1 in producing contraction on tissue strips and inducing calcium flux suggests that ET-1 might be an important mediator for modulating and maintaining corpus cavernosum smooth muscle tone. Therefore, additional exploration of the role of endothelins and their receptors in the tumescence and detumescence states of the penis would be extremely valuable. UROLOGY 60: 925–930, 2002. © 2002, Elsevier Science Inc.
N
ormal penile erection, a hemodynamic event involving both the central nervous system and local factors, is regulated by relaxation of the cavernosal arteries and corporal smooth muscle. Trabecular smooth muscle tone is tightly regulated by cholinergic, adrenergic, and nonadrenergicnoncholinergic (NANC) neurotransmitters.1–7 Studies have shown that after penile erection, the process of detumescence is regulated, at least in part, by the release of norepinephrine (NE) from the sympathetic nerves, resulting in activation of the alpha-adrenoreceptors in cavernosum tissue.8,9 From the Department of Reproductive Therapeutics, Johnson & Johnson Pharmaceutical Research & Development, Raritan, New Jersey Reprint requests: Harindra R. Abeysinghe, Ph.D., Johnson & Johnson Pharmaceutical Research & Development, Route 202, P.O. Box 300, Raritan, NJ 08869 Submitted: September 20, 2001, accepted (with revisions): May 16, 2002 © 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
However, it is not clear whether these nerves and signaling pathways are also responsible for providing constant tone to the cavernosal smooth muscle involved in maintaining penile flaccidity. Recently, attention has focused on the role of endothelin-1 (ET-1) as a potential modulator of corpus cavernosum basal tone and its importance in maintaining smooth muscle cell contractility when the penis is in the flaccid state. Studies have implicated ET-1 as a potent agonist for vascular smooth muscle cell contraction.10 –13 ET-1 is a 21-amino acid peptide secreted from the endothelial cells that line the walls of vascular tissue.14 –16 Similar to other vasoconstrictors, the physiologic effects of ET-1 are mediated by increasing intracellular calcium concentrations in cells. It is believed that, when released from endothelial cells into the surrounding tissue, ET-1 binds its receptor at the target cells and facilitates an increase in calcium by the release from intracel0090-4295/02/$22.00 PII S0090-4295(02)01845-9 925
lular calcium stores using the inositol triphosphate pathway and by direct influx using voltage-gated calcium channels.17–19 In human corpus cavernosum smooth muscle, ET-1 has been shown to elevate cytosolic and nuclear calcium levels in cells.10,20,21 The elevation of nuclear calcium is believed to play a role in inducing the expression of some of the immediate early genes such as c-myc and c-fos in human corpus cavernosum smooth muscle cells.21 It has also been demonstrated that ET-1 can increase cell proliferation in culture, which could be abrogated by blocking ET-1 receptors, as well as calcium channels.21 These observations suggest that ET-1 may be involved in regulating multiple physiologic pathways that are intimately linked to intracellular calcium regulation. In separately published reports, Christ et al.10 and Zhao and Christ20 have shown ET-1 to be a potent agonist for smooth muscle contraction in tissue, as well as for calcium flux in cells from corpus cavernosum. However, no direct comparison has been made for the effect of ET-1 on these two physiologic parameters. The purpose of this study was to investigate further the role of ET-1 and its receptor activity in cavernosal tissue and cells by measuring the physiologic responses to ET-1. For tissue studies, rabbit corporal tissue was excised, and tissue contraction in response to ET-1 and other agonists was measured in organ chambers. For cellular studies, primary myocyte cultures were established from rabbit corporal tissue, and changes in intracellular calcium levels induced by ET-1 were measured using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader (FLIPR). MATERIAL AND METHODS Corpus cavernosum tissue was obtained from sexually mature male New Zealand white rabbits. The animals were killed with a lethal injection of pentobarbital, and radical penectomy was performed. Rabbit corpus cavernosum smooth muscle cells were cultured following the procedures of Moreland et al.,22 with minor modifications.23 In brief, corporal tissue sections were washed several times in Hank’s balanced salt solution and then placed into a 10-cm tissue culture dish containing Dulbecco’s modified Eagles medium. The tissue pieces were mechanically separated and minced using a sharp scalpel. Approximately 1-mm3 pieces were transferred to six-well cluster plates, and cells growing out from the explants were cultured.
IMMUNOCYTOCHEMISTRY FOR SMOOTH MUSCLE CELL PROTEINS Cells were grown on coverslips, fixed, permeabilized, and stained with anti-desmin II mouse monoclonal antibody (ICN Pharmaceuticals, Aurora, Ohio) and anti-alpha-actin, smooth muscle mouse monoclonal antibodies (Calbiochem, San Diego, Calif), and visualized by fluorescence microscopy. 926
MEASUREMENT OF INTRACELLULAR CALCIUM FLUX USING FLIPR Intracellular calcium flux was measured using a FLIPR 384 following the manufacturer’s instructions (Molecular Devices, Sunnyvale, Calif). Corpus cavernosum cells were seeded into black-walled clear-base 96-well plates at a density of 20,000 cells per well. The next day, the medium was removed, and the cells were incubated for 1 hour at 37°C in the calcium chelating dye fluo-3AM (Molecular Probes, Eugene, Ore) mixed with a buffer solution. The dye solution was removed, and the cells were washed twice with buffer solution. ET-1, NE, and phenylephrine (PE, Sigma-Aldrich, St. Louis, Mo) were mixed with the buffer solution at the desired concentrations and separated into aliquots in triplicate into a 96-well compound plate. The 96-well plates were loaded into the FLIPR, and fluorescence readings were obtained at timed intervals. Liquid additions were made with an automated 96-channel pipetter in the FLIPR.
RABBIT CORPUS CAVERNOSUM TISSUE TENSION STUDIES Rabbit corpus cavernosum tissue was carefully dissected free from the surrounding tunica albuginea and cut into strips of approximately 0.3 ⫻ 0.3 ⫻ 1.2 cm3 and bathed in Krebs solution. The tissue strips were vertically mounted in organ baths with one end attached to an isometric transducer and the other to a glass hook at the bottom of the bath. The tissue strips were immersed in warmed (37°C) and oxygenated (95% oxygen plus 5% carbon dioxide) Krebs solution. Varying concentrations of ET-1 and PE were added directly to the organ baths, and the changes in isometric tension were recorded on a computer. The tissue tension measurements for each respective treatment of ET-1 and PE were performed on separate tissue strips, and the mean contraction for each treatment is reported from three separate contractions.
RESULTS Rabbit corpus cavernosum cells were characterized by immunofluorescence staining for muscle cell-specific filament proteins, desmin and alphaactin. The cells displayed intense longitudinal staining for both filament proteins (Fig. 1). It was estimated that between 90% and 95% of cells stained positive for both muscle cell markers. The addition of ET-1 to corpus cavernosum smooth muscle cells resulted in a transient increase of intracellular calcium (Fig. 2). The increases in calcium were dose dependent for ET-1, with a 100-nM concentration producing the maximal increase in fluorescence intensity. Treatment concentrations greater than 100 nM did not show a significant increase above the peak fluorescence for 100-nM ET-1 (data not shown). The lowest concentration of ET-1 capable of eliciting a calcium response was 5 nM. ET-1-induced calcium responses were completely abolished when the cells were treated with the ET-1 receptor antagonist PD145065 (data not shown). The conventional adrenoreceptor agonists NE and its synthetic analog PE were applied at different concentrations to the rabbit corpus cavernosum smooth muscle cells to determine the relative changes of intracellular calcium (individual fluoUROLOGY 60 (5), 2002
A
B
FIGURE 1. Immunocytochemistry on rabbit corpus cavernosum smooth muscle cell cultures. Cells were seeded onto coverslips and incubated with monoclonal antibodies recognizing (A) mouse desmin II and (B) smooth muscle alpha-actin followed by staining with fluorescein-conjugated antibodies to mouse IgG. Positive staining for actin and desmin intermediate filament proteins are clearly visible in cells.
FIGURE 2. ET-1 induction of a dose-dependent calcium response in corpus cavernosum cells. Results show fluorescence measurements taken from a single 96-well plate in which ET-1 was added to the cells at the indicated concentrations after 10 seconds. For the control, a similar volume of vehicle used to dissolve ET-1 was added to the cells. The data for each response curve are from a single representative experiment that was repeated three times.
rescence graphs not shown). A comparison of the calcium responses elicited by the different agonists ET-1, NE, and PE in the rabbit corpus cavernosum cells are profiled in Figure 3. The peak fluorescence unit recorded in response to each of the respective agonists is reported as a percentage of the UROLOGY 60 (5), 2002
maximal ET-1 response at the 100-nM dose. Treatment of cells with NE showed a dose-responsive calcium increase, with the maximal increases in cellular calcium occurring at 6 mM. The minimal concentration of NE required to elicit a measurable calcium response was 0.5 mM. For PE treatments 927
FIGURE 3. Comparison of the dose-response changes for intracellular calcium induced by the different agonists. Columns represent the percentage of fluorescence relative to a control treatment of 100 nM ET-1. ET-1 was clearly the most potent of the three agonists with 1 ⫻ 105 and 1 ⫻ 106 greater potency (at direct molar comparisons) than NE or PE, respectively.
at a similar concentration range, the calcium responses were significantly decreased. ET-1 produced concentration-dependent contractions in corpus cavernosum tissue when added to the organ baths (Fig. 4). Concentrations as low as 10 nM ET-1 were sufficient to increase tension more than twofold over baseline. A maximal contraction of almost eightfold over baseline was achieved at the 100-nM ET-1 dose. A 10-M dose of PE was required to elicit a muscle contraction of similar magnitude. PE at 1 M produced a contraction that was almost one half that, and a 100-nM dose did not produce any contraction of the tissue. The maximal contraction was achieved at the 10-M dose of PE; concentrations greater than this did not increase the amplitude of the contraction (data not shown). COMMENT The results of the present study demonstrate that ET-1 is a potent agonist for smooth muscle contraction in rabbit corpus cavernosum. The focus of this study was to examine some of the physiologic effects of ET-1 on corpus cavernosum cells and tissue. Interest has been growing in the potential role of ET-1 in modulating smooth muscle tone 928
when the penis is in the flaccid state. It has been widely speculated that a nonadrenergic mechanism may be involved in maintaining basal tone when the penis is in a flaccid state.10 –13 The results of our study demonstrate that transient increases of intracellular calcium are potentiated by ET-1 at concentrations as low as 10 nM. Increased calcium flux was dose dependent, with maximal increases seen at the 100-nM concentration level. To achieve a similar level of calcium flux in these rabbit corpus cavernosum cells, the adrenoreceptor agonists such as noradrenaline (NE) and its synthetic analog PE had to be applied at concentrations of 1 mM and greater. This observation is in accordance with previous reports indicating that ET-1 is considerably more potent than NE on a molar basis.6,13 However, our results clearly demonstrate that ET-1 is approximately 100,000 times more potent than NE and 1,000,000 times more potent than PE (by direct comparison of molar concentrations) in its ability to elicit a robust calcium response in the cavernosum cells. We discovered that to elicit a robust calcium response in the corpus cavernosum cells, the adrenoreceptor agonists had to be applied at very high concentrations, well above physiologic levels. A UROLOGY 60 (5), 2002
FIGURE 4. Changes in tissue tension for rabbit corpus cavernosum in response to contractile agonists and antagonists. Contraction and relaxation of tissue is presented in grams of tension. Basal tension for each of the tissues was adjusted such that all tissues began at approximately 1 g. Dose-response contraction was observed when tissues were treated with different concentrations of ET-1. Similar levels of contraction were achieved with 10 M PE and 100 nM ET-1.
possible explanation for the dramatic difference in calcium responses between NE and ET-1 in these cells is that ET-1 may be a primary mediator for modulating smooth muscle cell contraction and maintaining basal tone. This is supported by the data derived from the tissue tension studies and the calcium flux studies. The corpus cavernosum tissue responds with a high degree of sensitivity to PE at a concentration of 10 M, resulting in robust contractile activity. However, when cultured cells are treated with PE at this concentration range, no changes in intracellular calcium were detectable. It has been well documented that smooth muscle contraction, like all muscle, requires an increase of intracellular calcium to propagate a contraction.24 As shown in this study, to elicit a significant and detectable calcium flux in the cavernosum cells, the concentration levels of the adrenoreceptor agonists had to be augmented. However, as mentioned previously, the concentration of PE required to elicit a significant corporal smooth muscle contraction in muscle strips was 10 M, almost 100fold less. Intact corporal tissue strips, unlike cells in culture, consist of a heterogeneous mixture of corporal smooth muscle cells, vascular smooth UROLOGY 60 (5), 2002
muscle cells, endothelial cells, and fibroblasts. All these cell types together form an integral component of the cavernosum and have important structural and functional roles in this tissue. It is possible that the primary or immediate effect of PE is not directly on the corpus cavernosum smooth muscle cells, but instead, signals the release of other factors such as ET-1 from the endothelial cells. The local release of ET-1 then initiates the contraction of the cavernosal smooth muscle cells, thus maintaining the penis in a flaccid state. It is also plausible that PE and ET-1 may have a synergistic effect for the induction of smooth muscle contraction. This observation has been previously reported by Christ et al.10 from tissue tension studies on corporal tissue sections obtained from patients undergoing penile surgery. Their study revealed that coadministration of PE and ET-1 produced much greater contractile responses than those observed for PE alone. Regardless of whether ET-1 acts synergistically with other ligands or by itself, the results presented here clearly indicate that it is extremely potent in its ability to generate a calcium flux in cavernosum cells contraction. On the basis of published reports and our findings, it 929
seems plausible that ET-1 and ET-1 receptors may play an important physiologic role in corpus cavernosum smooth muscle function. CONCLUSIONS In this study, we found that ET-1 has a profound effect on corporal smooth muscle cell physiology in terms of its ability to both mobilize calcium and contract tissue rapidly. Its high potency compared with the adrenoreceptor agonists suggests that it may be an important mediator for maintaining smooth muscle cell basal tone in the penis. Additional studies to evaluate the in vitro and in vivo effects of ET-1 and their receptors is required to aid in the understanding of their precise physiologic role in corpus cavernosum smooth muscle contraction. These studies will also be beneficial in determining whether manipulation of ET-1 and its receptors will be useful for therapeutic purposes for male erectile dysfunction. ACKNOWLEDGMENT. To Dr. Joseph Gunnet for his assistance with the calcium imaging studies and Muh-Tsann Lai for technical assistance. REFERENCES 1. Adaikan PG, and Karim SMM: Adrenoreceptors in the human penis. J Auton Pharmacol 1: 199 –203, 1981. 2. Christ GJ, Maayani S, Valcic M, et al: Pharmacological studies of human erectile tissue: characteristics of spontaneous contractions and alterations in alpha-adrenoreceptor responsiveness with age and disease in isolated tissues. Br J Pharmacol 101: 375–381, 1990. 3. Diederichs W, Steif CG, Lue TF, et al: Norepinephrine involvement in penile detumescence. J Urol 143: 1264 –1266, 1990. 4. Hedlund H, and Andersson KE: Comparison of the responses to drugs acting on adrenoreceptors and muscarinic receptors in human isolated corpus cavernosum and cavernous artery. J Auton Pharmacol 5: 81–88, 1985. 5. Hedlund H, Andersson KE, and Mattiasson A: Pre- and post-junctional adreno- and muscarinic receptor function in isolated corpus spongiosum urethrae. J Auton Pharmacol 4: 241–246, 1984. 6. Saenz de Tejada I, Kim N, Lagan I, et al: Regulation of adrenergic activity in penile corpus cavernosum. J Urol 142: 1117–1121, 1989. 7. Saenz de Tejada I, Blanco R, Goldstein I, et al: Cholinergic neurotransmission in human corpus cavernosum. I. Responses of isolated tissue. Am J Physiol 254: 459 –467, 1988. 8. Krane RJ, Goldstein I, and Saenz de Tejada I: Impotence. N Engl J Med 321: 1648 –1659, 1989.
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