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Effects of noradrenaline on cytosolic concentrations of Ca2+ in cultured corpus cavernosum smooth muscle cells of the rabbit
Neuroscience Letters 324 (2002) 89–92 www.elsevier.com/locate/neulet
Effects of noradrenaline on cytosolic concentrations of Ca 21 in cultured corpus cavernosum smooth muscle cells of the rabbit Minoru Sato*, Masahito Kawatani Department of Physiology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan Received 20 October 2001; received in revised form 30 November 2001; accepted 12 December 2001
Abstract An immunohistochemical examination of cultured corpus cavernosum cells of the rabbit showed that they were composed of many smooth muscle cells retaining a-smooth muscle actin and a few of endothelial cells. Application of noradrenaline (NA, 0.5–100 mM) to the culture dose-dependently increased cytosolic concentrations of Ca 21 ([Ca 21]i) in smooth muscle cells. The NA (10 mM)-induced [Ca 21]i increase was dose-dependently inhibited by phenoxybenzamine (1 nM–1 mM). The inhibition was 71% at 0.1 mM and was completely made at 1 mM. An a1-adrenoceptor antagonist, prazosin (0.1 mM), inhibited the [Ca 21]i response by 42%, and propranolol (1 mM) enhanced the response by 118%. These adrenoceptor antagonists showed almost the same effects on a contractile response of corporal tissue strips to NA (10 mM) as the effects on the [Ca 21]i response. Our results suggest that NA-induced [Ca 21]i increase was mixed with a large increase through a1-adrenoceptors and a small decrease through b-adrenoceptors. The increased [Ca 21]i could play a role of intracellular messenger for leading to contraction of cavernosal smooth muscle cells. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: a-smooth muscle actin; Immunohistochemistry; Fura-2; Adrenoceptor; Phenoxybenzamine; Prazosin; Propranolol; Contraction
Adrenergic postganglionic nerve fiber excitation causes release of noradrenaline (NA) from the nerve terminals and elicites a contraction of corpus cavernosum smooth muscle, which leads to the detumescence of the erect penis [2]. Perfusion of the corpus cavernosum tissue strips with NA induced a contraction [2]. The NA-induced contraction was inhibited by an a-adrenoceptor antagonist (phenoxybenzamine, PBZ), an a1-adrenoceptor antagonist (prazosin) and a2-antagonists, and was enhanced by an b-adrenoceptor agonist [2]. However, signalling pathway underlying contraction mechanism induced by NA in corpus cavernosum smooth muscle cells remains to be solved. Changes in cytosolic concentrations of Ca 21 ([Ca 21]i) have been thought a second messenger in smooth muscle contraction [6]. In contractile experiments, the tissues contained smooth muscle cells and endothelial cells [7]. [Ca 21]i measurement experiments using dissociated corporal tissue cells have a benefit to examine whether NA has different effects in each type of the cells. It has been reported that an a-adrenoceptor agonist induced an increase of [Ca 21]i and contraction in * Corresponding author. Tel./fax: 181-18-836-2605. E-mail address: [email protected] (M. Sato).
corporal tissues of the rabbit [8]. One dose of a1-adrenoceptor agonist (100 mM) increased [Ca 21]i in cultured human cavernosal smooth muscle cells with a non-perfusing system [3]. We examined the effects of NA on [Ca 21]i in immunohistochemically-identified smooth muscle cells within cultured rabbit corpus cavernosum cells using fura-2AM microfluorometry. We compared the effects of adrenoceptor antagonists between the [Ca 21]i response of the cells to NA and a contractile response of the tissue strips to NA. A part of the present study has been reported in a preliminary form [11]. Male Japanese white rabbits (2.5–3.5 kg) were anesthetized with urethane (0.5 g/kg) by intraperitoneal injection. Penises were surgically and rapidly removed at the level of attachment of corporal body to the ischum and placed in a HEPES-buffered saline (HBS; 135.5 mM NaCl, 5.9 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2·6H2O, 11.6 mM glucose and 10 mM HEPES-NaOH, pH 7.4) which was bubbled with 100% O2. The corpus cavernosal tissues were carefully dissected free from the surrounding tunica albuginea. A part of them was cut into small pieces for culture and the most were longitudinaly sectioned into strips preparations (1.5 £ 1.5 £ 7 mm) for tension measurements.
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 57 9- 4
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M. Sato, M. Kawatani / Neuroscience Letters 324 (2002) 89–92
Culture of corpus cavernosal cells was performed by a modification of methods as described previously [10]. Small pieces of the tissue were incubated in 0.1% trypsin (Difco, Detroit, USA) and 0.2% collagenase (type II, Sigma, St. Louis, USA) containing HEPES-buffered minimum essential medium (MEM; Nissui, Tokyo, Japan; pH 7.4) at 378C for 30 min. After incubation, the pieces were rinsed with HEPES-buffered MEM and then mechanically dispersed with a Pasteur pipette (tip inside diameter 0.5 mm) in the medium. Dissociated cells were collected by centrifugation at 150 g for 5 min and resuspended in MEM supplemented with 10% fetal bovine serum (Intergen, Purchase, USA) and sodiumbicarbonate. The cells were seeded on collagencoated glass coverslips and cultured at 378C in a humidified atmosphere composed of 95% air and 5% CO2 for 5–10 days. An immunohistochemical examination of cultured corpus cavernosum cells was performed by an immunofluorescent staining method using fluorescein isothiocyanate-conjugated mouse monoclonal anti-a-smooth muscle actin antibody (1:500) (Sigma; St. Louis, USA) [12]. The cultured cavernosal cells were fixed with 4% formaldehyde in a phosphate-buffered saline (PBS; 10 mM, pH 7.4), permeabilized with 0.5% Triton X-100 in PBS and washed with 1% bovine serum albumin in PBS. The cells were incubated with the antibody for 30 min at room temperature. After washed with PBS, the cells were covered with glycerolcontaining PBS (1:3) and visualized by a fluorescent microscope (Olympus BH2, Tokyo, Japan). Measurements of [Ca 21]i were performed by microfluorometry using fura-2AM [5] in accordance with previous reports [10]. Briefly, the coverslips with cultured cells were washed with HBS and loaded with 5 mM fura-2AM (Dojindo Lab., Kumamoto, Japan) in HBS at 22–278C for 1 h. Fluorescence was generated by double-beam excitation (340 and 360 nm). The fluorescent images of the cells were displayed and analyzed with a video-frame memory system equipped with a computerized image-analysis unit. [Ca 21]i was determined by reference to a calibration curve obtained in vitro. A chemical solution was introduced into the bath in which cultured cavernosal cells were being perfused. The replacement of the bath solution was carried out within 30 s.
An increase in [Ca 21]i was calculated as the difference between the level of [Ca 21]i during application of NA and a resting level. Contractile tension of the corpus cavernosum strips were isometrically measured by a force transducer (Type 45196A; GE Marquette Medical Sys. Japan Ltd., Tokyo, Japan) with an amplyfier (Type 1829; GE Marquette Medical Sys. Japan Ltd.) and recorded on a pen recorder (R-62; Rikadenki Kogyo CO, Ltd., Tokyo, Japan). The tissue strips were placed vertically in a thermostatically controlled (378C) organ bath (10 ml) which was filled with 100% O2bubbled HBS. The tension of the strips was adjusted to 0.2– 0.4 g (a basal tension) and allowed to equilibrate for 2–3 h before experiments. Adrenoceptor antagonists were injected to the in vitro preparations prior to NA application. The inhibitions by antagonists were calculated as test/control and expressed as percentage. All averaged data have been expressed as mean ^ standard error of the mean (SEM). Statistical analysis was performed by the Mann–Whitney test and paired ttest. P , 0:05 was used as an indicator of statistical significance. PBZ and prazosin were purchased from Nakarai (Kyoto, Japan) and Sigma (St. Louis, USA), respectively. All other chemicals were obtained from Wako (Oosaka, Japan). The chemicals were added to the HBS before experiments. An immunohistochemical examination of cultured corpus cavernosum cells showed that they were composed of many smooth muscle cells, which contained anti-a-smooth muscle actin antibody-positive stress fibers (Fig. 1A). A few of stratified cobblestone morphologies which are characteristic of endothelial cells was not stained with the fluorescence (Fig. 1B). Application of NA (0.5–100 mM) induced a dose-dependent increase of [Ca 21]i in cultured corpus cavernosum smooth muscle cells (Fig. 2). The NA effect became detectable within 1 min after the application. After the removal of NA, [Ca 21]i returned to a resting level within 1–2 min. The threshold dose of the [Ca 21]i response was 0.5 mM, at which [Ca 21]i increased from a resting level of 74 ^ 3 to 124 ^ 9 nM (154 ^ 12%; n ¼ 6, P , 0:05) in 12% of the cells (Fig. 2). At 10 mM, [Ca 21]i increased to 319 ^ 20 nM
Fig. 1. An immunofluorescence photomicrograph of cultured (5–10 days) corpus cavernosum cells of the rabbit. (A) Many of the cultured cells have typical patterns of F-actin stress fibers (arrows) which are heavily stained with dye-conjugated anti-a-smooth muscle actin antibody. (B) Endothelial cells are not stained with the dye. Bar represents 50 mm.
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Fig. 2. A dose-dependent response curve of cytosolic concentrations of Ca 21 ([Ca 21]i) to NA in cultured corpus cavernosum smooth muscle cells. NA was applied from 0.01 to 100 mM.
corpus cavernosum endothelial cells exhibited no response of [Ca 21]i to NA. Accordingly, endothelin-1 might not be involved in the mechanism for NA-induced smooth muscle contraction. NA-induced [Ca 21]i increase in cultured cavernosal smooth muscle cells was inhibited by PBZ or prazosin (Fig. 3). This suggests that the [Ca 21]i increase was mediated by a-adrenoceptors including a1-type. The present contractile response data and previous reports showed that NA-induced contraction of cavernosal tissue strips was inhibited by a-adrenoceptor antagonists [2]. Furthermore, an a1-agonist induced [Ca 21]i increase in cultured human corpoal smooth muscle cells [3]. Since a1-agonist-induced cavernosal smooth muscle contraction
(317 ^ 33%; n ¼ 21, P , 0:001) in 38% of the cells. The changes were not different from the [Ca 21]i response to 10 mM NA in the smooth muscle cells co-cultured with endothelial cells. No response of [Ca 21]i to NA (0.5–100 mM) was seen in the cultured corpus cavernosum endothelial cells. During application of 100 mM NA, [Ca 21]i did not alter (91 ^ 5 nM, 3 ^ 1%; n ¼ 6, P . 0:05) from a resting level (88 ^ 5 nM). To asses which type of adrenoceptors are involved in the [Ca 21]i response to NA in cultured cavernosal smooth muscle cells, the effects of a- and b-adrenoceptor antagonists on the [Ca 21]i response were tested. In the presence of 1 mM PBZ, 10 mM NA did not alter [Ca 21]i from a resting level (Fig. 3A). The inhibitory effect of PBZ was dosedependent from 1 nM to 1 mM (Fig. 3D). PBZ, at 0.1 mM, inhibited the NA-induced [Ca 21]i increase by 71 ^ 5% and, at 1 mM, abolished. In the presence of 0.1 mM prazosin, an a1-adrenoceptor antagonist, the [Ca 21]i increase (284 ^ 36 nM) was inhibited by 42 ^ 9% (197 ^ 25 nM, n ¼ 4, P , 0:05) (Fig. 3C). Propranolol (PPL), at 1 mM, augmented the NA-induced [Ca 21]i increase (243 ^ 29 nM) by 118 ^ 3% (282 ^ 27 nM, n ¼ 4, P , 0:05) (Figs. 3C and D). Fig. 4 shows the effects of adrenoceptor antagonists on a contractile response to NA in corpus cavernosum tissue strips. PBZ (0.1 nM–10 mM) and prazosin (1 nM–10 mM) depressed 10 mM NA-induced contraction in a dose-dependent manner. The degree of the inhibition by 10 nM PBZ (59 ^ 12%, n ¼ 5, P , 0:05) was higher than that by 10 nM prazosin (30 ^ 8%, n ¼ 5, P , 0:05). PPL, at 10 mM, slightly enhanced the contraction (111 ^ 6%, n ¼ 5, P , 0:05). The present study directly demonstrated that NA increased [Ca 21]i in cultured cavernosal smooth muscle cells of the rabbit. The [Ca 21]i increase and NA-induced cavernosal strips contraction were inhibited by a-adrenoceptor antagonists including an a1-antagonist and were slightly augmented by an b-antagonist. It has been known that endothelin-1 which secrets from endothelial cells elicited both corporal smooth muscle contraction and [Ca 21]i increase in the tissue smooth muscle cells [2,15]. But the present data showed that cultured
Fig. 3. Effects of adrenoceptor antagonists on NA-induced [Ca 21]i increase in cultured cavernosal smooth muscle cells. Representative effects of PBZ, PPL on NA-induced [Ca 21]i increase were shown in (A), (B) and (C), respectively. A thick bar indicates the duration of NA (10 mM) application. Thin bars indicate the duration of antagonists. After an increase in [Ca 21]i was induced by the first NA application (left of all), PBZ (1 mM), prazosin (0.1 mM) and PPL (1 mM) were administrated to cultured cells 1–2 min before the second NA application. Test was finished by washing out the drugs with a perfusing medium. Mean of the two [Ca 21]i responses to NA before and after test was used as control. Even after removal of PBZ from a perfusion medium, [Ca 21]i response to NA did not recover due to an irreversible effect of PBZ. (D) Dose-dependent effects of PBZ (0.1 nM–1 mM) and PPL (0.1–10 mM) on 10 mM NA-induced [Ca 21]i increase.
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decrease of [Ca 21]i in cultured cavernosal smooth muscle cells. The present data agree with a report that PPL potentiated the contractile action of NA in human corpus cavernosum [1]. The putative decrease in [Ca 21]i in response to NA might be caused by b-adrenoceptors which activates cAMP/Ca 21 extrusion pathway [6].
Fig. 4. Effects of adrenoceptor antagonists on NA-induced cavernosal strips contraction. Dose-dependent effects of PBZ (0.1 nM– 10 mM), prazosin (1 nM–10 mM) and PPL (0.1–10 mM) on 10 mM NA-induced contraction were shown.
of the human and rabbit was mediated by a1A- and a1Badrenoceptor subtypes [4,13], the subtypes might be involved in the present NA-mediated [Ca 21]i increase. Whereas the amplitude of the inhibition of NA-induced [Ca 21]i increase by 0.1 mM PBZ was about 71%, the amplitude of that by 0.1 mM prazosin was about 42%. The different inhibitory efficacy between a- and a1-antagonists was also seen in the present contractile response (Fig. 4). These suggest that a2-adrenoceptor other than a1-adrenoceptor could be partly involved in the NA-induced [Ca 21]i increase. This finding is supported by reports that a2agonists have evoked contractile response of corpus cavernosal smooth muscle [2]. Contraction of corporal smooth muscle induced by aadrenoceptor agonist was dependent on both intra- and extracellular calcium [2], and microinjection of inositol 1,4,5-triphosphate (IP3) into rabbit corporal smooth muscle cells produced an increase in [Ca 21]i [3]. In vascular smooth muscle, stimulation of a1-adrenoceptors forms IP3 causing release of Ca 21 from intracellular stores [9], and a2-adrenoceptors-mediated contraction requires an influx of extracellular Ca 21 [14]. From these findings, an increase in [Ca 21]i induced by NA might be due to activation of both Ca 21 release system and Ca 21 influx system in corpus cavernosal smooth muscle cells. It is needed to further study for determination of a1- and a2-adrenoceptors mediating [Ca 21]i increase systems which were responsible to NA in our preparations. Both NA-induced increases in [Ca 21]i and contractile tension were slightly enhanced by PPL. Thus, stimulation by NA of b-adrenoceptors which occupies 10% of adrenoceptors in corpus cavernosum tissues [2] could cause a
[1] Adaikan, P.G. and Karim, S.M.M., Adrenoceptors in the human penis, J. Auton. Pharmacol., 1 (1981) 199–203. [2] Andersson, K.-E. and Wagner, G., Physiology of penile erection, Physiol. Rev., 75 (1995) 191–236. [3] Christ, G.J., Moreno, A.P., Melman, A. and Spray, D.C., Gap junction-mediated intercellular diffusion of Ca 21 in cultured human corporal smooth muscle cells, Am. J. Physiol., 263 (1992) C373–C383. [4] Furukawa, K., Chess-Williams, R. and Uchiyama, T., a1Badrenoceptor subtype mediating the phenylephrineinduced contractile response in rabbit corpus cavernosum penis, Jpn. J. Pharmacol., 71 (1996) 325–331. [5] Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 21 indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440–3450. [6] Horowitz, A., Menice, C.B., Lapoprte, R. and Morgan, K.G., Mechanisms of smooth muscle contraction, Physiol. Rev., 76 (1996) 967–1003. [7] Jevtich, M.J., Khawand, N.Y. and Vidic, B., Clinical significance of ultrastructual findings of the corpus cavernosum of normal and impotent men, J. Urol., 143 (1990) 289–292. [8] Levin, R.M., Tong, Yat-Ching, Hypolite, J.A. and Broderick, G., Correlation of calcium movements with the contractile response of the rabbit corpus cavernosum using fura-2 calcium fluorescence, Int. J. Impot. Res., 3 (1991) 141–153. [9] Minneman, K.P., a1-adrenergic receptor subtypes, inositol triphosphate and sources of cell Ca 21, Pharmacol. Rev., 40 (1988) 87–119. [10] Sato, M. and Kawatani, M., Nitric oxide raises cytosolic concentrations of Ca 21 in cultured nodose ganglion neurons from rabbits, Neurosci. Lett., 206 (1996) 69–72. [11] Sato, M. and Kawatani, M., Effects of noradrenaline on cytosolic calcium in cultured corpus cavernosum smooth muscle cells of the rabbit, Abstr. Soc. Neurosci., 24 (1998) 1620. [12] Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillenssen, D. and Gabbiani, G., A monoclonal antibody against asmooth muscle actin: a new probe for smooth muscle differentiation, J. Cell Biol., 103 (1986) 2787–2796. [13] Traish, A.M., Netsuwan, N., Daley, J., Padman-Nathan, H., Goldstein, I. and Saenz de Tejada, I., A heterogeneous population of a1 adrenergic receptors mediates contraction of human corpus cavernosum smooth muscle to norepinephrine, J. Urol., 153 (1995) 222–227. [14] Van Meel, J.C., De Jonge, A., Kalkman, H.O., Wilffert, B., Timmermans, P.B.M.W.M. and Van Zwieten, P.A., Vascular smooth muscle contraction initiated postsynaptic alpha 2adrenoceptor activation is induced by an influx of extracellular calcium, Eur. J. Pharmacol., 69 (1981) 205–208. [15] Zhao, W. and Christ, G.T., Endothelin-1 as a putative modulator of an erectile dysfunction. II. Calcium mobilization in cultured human corporal smooth muscle cells, J. Urol., 154 (1995) 1571–1579.
Effects of noradrenaline on cytosolic concentrations of Ca 21 in cultured corpus cavernosum smooth muscle cells of the rabbit Minoru Sato*, Masahito Kawatani Department of Physiology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan Received 20 October 2001; received in revised form 30 November 2001; accepted 12 December 2001
Abstract An immunohistochemical examination of cultured corpus cavernosum cells of the rabbit showed that they were composed of many smooth muscle cells retaining a-smooth muscle actin and a few of endothelial cells. Application of noradrenaline (NA, 0.5–100 mM) to the culture dose-dependently increased cytosolic concentrations of Ca 21 ([Ca 21]i) in smooth muscle cells. The NA (10 mM)-induced [Ca 21]i increase was dose-dependently inhibited by phenoxybenzamine (1 nM–1 mM). The inhibition was 71% at 0.1 mM and was completely made at 1 mM. An a1-adrenoceptor antagonist, prazosin (0.1 mM), inhibited the [Ca 21]i response by 42%, and propranolol (1 mM) enhanced the response by 118%. These adrenoceptor antagonists showed almost the same effects on a contractile response of corporal tissue strips to NA (10 mM) as the effects on the [Ca 21]i response. Our results suggest that NA-induced [Ca 21]i increase was mixed with a large increase through a1-adrenoceptors and a small decrease through b-adrenoceptors. The increased [Ca 21]i could play a role of intracellular messenger for leading to contraction of cavernosal smooth muscle cells. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: a-smooth muscle actin; Immunohistochemistry; Fura-2; Adrenoceptor; Phenoxybenzamine; Prazosin; Propranolol; Contraction
Adrenergic postganglionic nerve fiber excitation causes release of noradrenaline (NA) from the nerve terminals and elicites a contraction of corpus cavernosum smooth muscle, which leads to the detumescence of the erect penis [2]. Perfusion of the corpus cavernosum tissue strips with NA induced a contraction [2]. The NA-induced contraction was inhibited by an a-adrenoceptor antagonist (phenoxybenzamine, PBZ), an a1-adrenoceptor antagonist (prazosin) and a2-antagonists, and was enhanced by an b-adrenoceptor agonist [2]. However, signalling pathway underlying contraction mechanism induced by NA in corpus cavernosum smooth muscle cells remains to be solved. Changes in cytosolic concentrations of Ca 21 ([Ca 21]i) have been thought a second messenger in smooth muscle contraction [6]. In contractile experiments, the tissues contained smooth muscle cells and endothelial cells [7]. [Ca 21]i measurement experiments using dissociated corporal tissue cells have a benefit to examine whether NA has different effects in each type of the cells. It has been reported that an a-adrenoceptor agonist induced an increase of [Ca 21]i and contraction in * Corresponding author. Tel./fax: 181-18-836-2605. E-mail address: [email protected] (M. Sato).
corporal tissues of the rabbit [8]. One dose of a1-adrenoceptor agonist (100 mM) increased [Ca 21]i in cultured human cavernosal smooth muscle cells with a non-perfusing system [3]. We examined the effects of NA on [Ca 21]i in immunohistochemically-identified smooth muscle cells within cultured rabbit corpus cavernosum cells using fura-2AM microfluorometry. We compared the effects of adrenoceptor antagonists between the [Ca 21]i response of the cells to NA and a contractile response of the tissue strips to NA. A part of the present study has been reported in a preliminary form [11]. Male Japanese white rabbits (2.5–3.5 kg) were anesthetized with urethane (0.5 g/kg) by intraperitoneal injection. Penises were surgically and rapidly removed at the level of attachment of corporal body to the ischum and placed in a HEPES-buffered saline (HBS; 135.5 mM NaCl, 5.9 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2·6H2O, 11.6 mM glucose and 10 mM HEPES-NaOH, pH 7.4) which was bubbled with 100% O2. The corpus cavernosal tissues were carefully dissected free from the surrounding tunica albuginea. A part of them was cut into small pieces for culture and the most were longitudinaly sectioned into strips preparations (1.5 £ 1.5 £ 7 mm) for tension measurements.
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 57 9- 4
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Culture of corpus cavernosal cells was performed by a modification of methods as described previously [10]. Small pieces of the tissue were incubated in 0.1% trypsin (Difco, Detroit, USA) and 0.2% collagenase (type II, Sigma, St. Louis, USA) containing HEPES-buffered minimum essential medium (MEM; Nissui, Tokyo, Japan; pH 7.4) at 378C for 30 min. After incubation, the pieces were rinsed with HEPES-buffered MEM and then mechanically dispersed with a Pasteur pipette (tip inside diameter 0.5 mm) in the medium. Dissociated cells were collected by centrifugation at 150 g for 5 min and resuspended in MEM supplemented with 10% fetal bovine serum (Intergen, Purchase, USA) and sodiumbicarbonate. The cells were seeded on collagencoated glass coverslips and cultured at 378C in a humidified atmosphere composed of 95% air and 5% CO2 for 5–10 days. An immunohistochemical examination of cultured corpus cavernosum cells was performed by an immunofluorescent staining method using fluorescein isothiocyanate-conjugated mouse monoclonal anti-a-smooth muscle actin antibody (1:500) (Sigma; St. Louis, USA) [12]. The cultured cavernosal cells were fixed with 4% formaldehyde in a phosphate-buffered saline (PBS; 10 mM, pH 7.4), permeabilized with 0.5% Triton X-100 in PBS and washed with 1% bovine serum albumin in PBS. The cells were incubated with the antibody for 30 min at room temperature. After washed with PBS, the cells were covered with glycerolcontaining PBS (1:3) and visualized by a fluorescent microscope (Olympus BH2, Tokyo, Japan). Measurements of [Ca 21]i were performed by microfluorometry using fura-2AM [5] in accordance with previous reports [10]. Briefly, the coverslips with cultured cells were washed with HBS and loaded with 5 mM fura-2AM (Dojindo Lab., Kumamoto, Japan) in HBS at 22–278C for 1 h. Fluorescence was generated by double-beam excitation (340 and 360 nm). The fluorescent images of the cells were displayed and analyzed with a video-frame memory system equipped with a computerized image-analysis unit. [Ca 21]i was determined by reference to a calibration curve obtained in vitro. A chemical solution was introduced into the bath in which cultured cavernosal cells were being perfused. The replacement of the bath solution was carried out within 30 s.
An increase in [Ca 21]i was calculated as the difference between the level of [Ca 21]i during application of NA and a resting level. Contractile tension of the corpus cavernosum strips were isometrically measured by a force transducer (Type 45196A; GE Marquette Medical Sys. Japan Ltd., Tokyo, Japan) with an amplyfier (Type 1829; GE Marquette Medical Sys. Japan Ltd.) and recorded on a pen recorder (R-62; Rikadenki Kogyo CO, Ltd., Tokyo, Japan). The tissue strips were placed vertically in a thermostatically controlled (378C) organ bath (10 ml) which was filled with 100% O2bubbled HBS. The tension of the strips was adjusted to 0.2– 0.4 g (a basal tension) and allowed to equilibrate for 2–3 h before experiments. Adrenoceptor antagonists were injected to the in vitro preparations prior to NA application. The inhibitions by antagonists were calculated as test/control and expressed as percentage. All averaged data have been expressed as mean ^ standard error of the mean (SEM). Statistical analysis was performed by the Mann–Whitney test and paired ttest. P , 0:05 was used as an indicator of statistical significance. PBZ and prazosin were purchased from Nakarai (Kyoto, Japan) and Sigma (St. Louis, USA), respectively. All other chemicals were obtained from Wako (Oosaka, Japan). The chemicals were added to the HBS before experiments. An immunohistochemical examination of cultured corpus cavernosum cells showed that they were composed of many smooth muscle cells, which contained anti-a-smooth muscle actin antibody-positive stress fibers (Fig. 1A). A few of stratified cobblestone morphologies which are characteristic of endothelial cells was not stained with the fluorescence (Fig. 1B). Application of NA (0.5–100 mM) induced a dose-dependent increase of [Ca 21]i in cultured corpus cavernosum smooth muscle cells (Fig. 2). The NA effect became detectable within 1 min after the application. After the removal of NA, [Ca 21]i returned to a resting level within 1–2 min. The threshold dose of the [Ca 21]i response was 0.5 mM, at which [Ca 21]i increased from a resting level of 74 ^ 3 to 124 ^ 9 nM (154 ^ 12%; n ¼ 6, P , 0:05) in 12% of the cells (Fig. 2). At 10 mM, [Ca 21]i increased to 319 ^ 20 nM
Fig. 1. An immunofluorescence photomicrograph of cultured (5–10 days) corpus cavernosum cells of the rabbit. (A) Many of the cultured cells have typical patterns of F-actin stress fibers (arrows) which are heavily stained with dye-conjugated anti-a-smooth muscle actin antibody. (B) Endothelial cells are not stained with the dye. Bar represents 50 mm.
M. Sato, M. Kawatani / Neuroscience Letters 324 (2002) 89–92
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Fig. 2. A dose-dependent response curve of cytosolic concentrations of Ca 21 ([Ca 21]i) to NA in cultured corpus cavernosum smooth muscle cells. NA was applied from 0.01 to 100 mM.
corpus cavernosum endothelial cells exhibited no response of [Ca 21]i to NA. Accordingly, endothelin-1 might not be involved in the mechanism for NA-induced smooth muscle contraction. NA-induced [Ca 21]i increase in cultured cavernosal smooth muscle cells was inhibited by PBZ or prazosin (Fig. 3). This suggests that the [Ca 21]i increase was mediated by a-adrenoceptors including a1-type. The present contractile response data and previous reports showed that NA-induced contraction of cavernosal tissue strips was inhibited by a-adrenoceptor antagonists [2]. Furthermore, an a1-agonist induced [Ca 21]i increase in cultured human corpoal smooth muscle cells [3]. Since a1-agonist-induced cavernosal smooth muscle contraction
(317 ^ 33%; n ¼ 21, P , 0:001) in 38% of the cells. The changes were not different from the [Ca 21]i response to 10 mM NA in the smooth muscle cells co-cultured with endothelial cells. No response of [Ca 21]i to NA (0.5–100 mM) was seen in the cultured corpus cavernosum endothelial cells. During application of 100 mM NA, [Ca 21]i did not alter (91 ^ 5 nM, 3 ^ 1%; n ¼ 6, P . 0:05) from a resting level (88 ^ 5 nM). To asses which type of adrenoceptors are involved in the [Ca 21]i response to NA in cultured cavernosal smooth muscle cells, the effects of a- and b-adrenoceptor antagonists on the [Ca 21]i response were tested. In the presence of 1 mM PBZ, 10 mM NA did not alter [Ca 21]i from a resting level (Fig. 3A). The inhibitory effect of PBZ was dosedependent from 1 nM to 1 mM (Fig. 3D). PBZ, at 0.1 mM, inhibited the NA-induced [Ca 21]i increase by 71 ^ 5% and, at 1 mM, abolished. In the presence of 0.1 mM prazosin, an a1-adrenoceptor antagonist, the [Ca 21]i increase (284 ^ 36 nM) was inhibited by 42 ^ 9% (197 ^ 25 nM, n ¼ 4, P , 0:05) (Fig. 3C). Propranolol (PPL), at 1 mM, augmented the NA-induced [Ca 21]i increase (243 ^ 29 nM) by 118 ^ 3% (282 ^ 27 nM, n ¼ 4, P , 0:05) (Figs. 3C and D). Fig. 4 shows the effects of adrenoceptor antagonists on a contractile response to NA in corpus cavernosum tissue strips. PBZ (0.1 nM–10 mM) and prazosin (1 nM–10 mM) depressed 10 mM NA-induced contraction in a dose-dependent manner. The degree of the inhibition by 10 nM PBZ (59 ^ 12%, n ¼ 5, P , 0:05) was higher than that by 10 nM prazosin (30 ^ 8%, n ¼ 5, P , 0:05). PPL, at 10 mM, slightly enhanced the contraction (111 ^ 6%, n ¼ 5, P , 0:05). The present study directly demonstrated that NA increased [Ca 21]i in cultured cavernosal smooth muscle cells of the rabbit. The [Ca 21]i increase and NA-induced cavernosal strips contraction were inhibited by a-adrenoceptor antagonists including an a1-antagonist and were slightly augmented by an b-antagonist. It has been known that endothelin-1 which secrets from endothelial cells elicited both corporal smooth muscle contraction and [Ca 21]i increase in the tissue smooth muscle cells [2,15]. But the present data showed that cultured
Fig. 3. Effects of adrenoceptor antagonists on NA-induced [Ca 21]i increase in cultured cavernosal smooth muscle cells. Representative effects of PBZ, PPL on NA-induced [Ca 21]i increase were shown in (A), (B) and (C), respectively. A thick bar indicates the duration of NA (10 mM) application. Thin bars indicate the duration of antagonists. After an increase in [Ca 21]i was induced by the first NA application (left of all), PBZ (1 mM), prazosin (0.1 mM) and PPL (1 mM) were administrated to cultured cells 1–2 min before the second NA application. Test was finished by washing out the drugs with a perfusing medium. Mean of the two [Ca 21]i responses to NA before and after test was used as control. Even after removal of PBZ from a perfusion medium, [Ca 21]i response to NA did not recover due to an irreversible effect of PBZ. (D) Dose-dependent effects of PBZ (0.1 nM–1 mM) and PPL (0.1–10 mM) on 10 mM NA-induced [Ca 21]i increase.
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decrease of [Ca 21]i in cultured cavernosal smooth muscle cells. The present data agree with a report that PPL potentiated the contractile action of NA in human corpus cavernosum [1]. The putative decrease in [Ca 21]i in response to NA might be caused by b-adrenoceptors which activates cAMP/Ca 21 extrusion pathway [6].
Fig. 4. Effects of adrenoceptor antagonists on NA-induced cavernosal strips contraction. Dose-dependent effects of PBZ (0.1 nM– 10 mM), prazosin (1 nM–10 mM) and PPL (0.1–10 mM) on 10 mM NA-induced contraction were shown.
of the human and rabbit was mediated by a1A- and a1Badrenoceptor subtypes [4,13], the subtypes might be involved in the present NA-mediated [Ca 21]i increase. Whereas the amplitude of the inhibition of NA-induced [Ca 21]i increase by 0.1 mM PBZ was about 71%, the amplitude of that by 0.1 mM prazosin was about 42%. The different inhibitory efficacy between a- and a1-antagonists was also seen in the present contractile response (Fig. 4). These suggest that a2-adrenoceptor other than a1-adrenoceptor could be partly involved in the NA-induced [Ca 21]i increase. This finding is supported by reports that a2agonists have evoked contractile response of corpus cavernosal smooth muscle [2]. Contraction of corporal smooth muscle induced by aadrenoceptor agonist was dependent on both intra- and extracellular calcium [2], and microinjection of inositol 1,4,5-triphosphate (IP3) into rabbit corporal smooth muscle cells produced an increase in [Ca 21]i [3]. In vascular smooth muscle, stimulation of a1-adrenoceptors forms IP3 causing release of Ca 21 from intracellular stores [9], and a2-adrenoceptors-mediated contraction requires an influx of extracellular Ca 21 [14]. From these findings, an increase in [Ca 21]i induced by NA might be due to activation of both Ca 21 release system and Ca 21 influx system in corpus cavernosal smooth muscle cells. It is needed to further study for determination of a1- and a2-adrenoceptors mediating [Ca 21]i increase systems which were responsible to NA in our preparations. Both NA-induced increases in [Ca 21]i and contractile tension were slightly enhanced by PPL. Thus, stimulation by NA of b-adrenoceptors which occupies 10% of adrenoceptors in corpus cavernosum tissues [2] could cause a
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