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K+-ATPase pump and Ca2+-activated K+ channels
Life Sciences 83 (2008) 438–446
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Airway smooth muscle relaxation induced by 5-HT2A receptors: Role of Na+/K+-ATPase pump and Ca2+-activated K+ channels Patricia Campos-Bedolla a, Mario H. Vargas b, Patricia Segura b, Verónica Carbajal b, Eduardo Calixto c, Alejandra Figueroa e, Edgar Flores-Soto e, Carlos Barajas-López d, Nicandro Mendoza-Patiño e, Luis M. Montaño e,⁎ a
Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México DF, Mexico Departamento de Investigación en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, México DF, Mexico c Departamento de Neurobiología, División de Investigación en Neurociencias, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, México DF, Mexico d División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, SLP, Mexico e Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, México DF, Mexico b
a r t i c l e
i n f o
Article history: Received 14 February 2008 Accepted 17 July 2008 Keywords: Airway smooth muscle Relaxation 5-HT2A receptors BKCa channels Na+/K+-ATPase pump
a b s t r a c t Aims: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. Main methods: Using organ baths, patch clamp, and intracellular Ca2+ measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. Key findings: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 μM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 μM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT1A, 5-HT1B/5-HT1D and 5-HT2B receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 μM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K+ currents (IK(Ca)), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. Significance: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na+/K+-ATPase pump, and increasing activity of the high-conductance Ca2+-activated K+ channels, probably via 5-HT2A receptors. © 2008 Elsevier Inc. All rights reserved.
Introduction 5-Hydroxytryptamine (5-HT or serotonin) is an important neurotransmitter of the central nervous system and the digestive tract, and has a major role in some conditions such as migraine and inflammatory bowel disease. Serotonergic fibers have not been described in the respiratory system, but in different mammalian species, including humans, there are non-neuronal sources of 5-HT such as mast cells and neuroendocrine cells (Joos et al., 1997; Lauweryns et al., 1974; Fu et al., 2002). Moreover, in pulmonary tissue, 5-HT levels are directly proportional to their plasmatic ⁎ Corresponding author. Departamento de Farmacología, Edificio de Investigación, sexto piso, laboratorio 3, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510, México DF, Mexico. Tel./fax: +55 56665868. E-mail address: [email protected] (L.M. Montaño). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.07.006
concentrations, being the platelets as its main source (Cazzola and Matera 2000). The role of 5-HT in asthma has been controversial, but there are some clues indicating its potential involvement in this disease. Plasma concentration of free 5-HT notably increases during an asthmatic exacerbation and this increment is related to asthma severity (Lechin et al., 1996). In addition, it has been reported that tianeptine (a drug that lowers plasma 5-HT by enhancing the 5-HT re-uptake) improves pulmonary function in asthmatic children (Lechin et al., 1998). Therefore, it is important to better understand the physiologic effect of 5-HT on the airway smooth muscle. 5-HT induces a direct sustained contraction of airway smooth muscle from bovine, dog, equine or mouse (Goldie et al., 1982; Lemoine and Kaumann 1986; Doucett et al., 1990; Buckner et al., 1991; Baron et al., 1993; Adner et al., 2002), but in other preparations such as guinea pig trachea or human bronchi the responses elicited by 5-HT
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are biphasic (contraction followed by relaxation) in nature (Goldie et al., 1982; Baumgartner et al., 1990; Ben-Harari et al., 1994). The relaxing phase of this biphasic response has been proposed to occur only at 5-HT concentrations ≥10 µM (Baumgartner et al., 1990), and it has been postulated that 5-HT2 receptor is the main serotonergic receptor involved in this effect. Further characterization of the mechanisms involved in the 5-HT-induced relaxation has been scarcely investigated. Baumgartner et al. (1990) described that the relaxation caused by high 5-HT concentrations in guinea pig tracheal strips was coincident with a decrease of IP3 production, and they postulated that an increase in the cAMP might be involved. By evaluating the ouabain-sensitive 86Rb+ uptake in cultured guinea pig tracheal smooth muscle cells, Rhoden et al. (2000) found that 5-HT stimulated the activity of Na+/K+-ATPase via 5-HT2A receptors, but these authors did not explore the physiological consequences of such stimulation. Finally, Ben-Harari et al. (1994) postulated that the relaxation phase induced by a single concentration of 5-HT was related to a receptor-dependent desensitization. The present work was aimed to investigate the potential role of several relaxing mechanisms triggered by 5-HT in guinea pig airway smooth muscle, including the role of Na+/K+-ATPase and highconductance Ca2+-activated K+ (BKCa) channels. Materials and methods Animals Male Hartley guinea pigs (500–600 g) bred in conventional conditions in our institutional animal facilities (filtered conditioned air, 21 ± 1 °C, 50–70% humidity, sterilized bed) and fed with Harlan® pellets and sterilized water were used. The protocol was approved by the Scientific and Bioethics Committees of the Instituto Nacional de Enfermedades Respiratorias. The experiments were conducted in accordance with the published Guiding Principles in the Care and Use of Animals, approved by the American Physiological Society. Sensitization procedure and antigenic challenge Guinea pigs were sensitized at day 0 by intraperitoneal administration of 60 mg ovalbumin (OA) and 1 mg Al(OH)3 in 0.5 ml of saline (0.9% NaCl). At day 8, the animals were nebulized with 3 mg·ml− 1 OA in saline for 5 min, delivered by a ultrasonic nebulizer (model US-1, Puritan Bennett, Carlsbad, CA). Guinea pigs were nebulized again on day 15 with 0.5 mg·ml− 1 OA in saline for 1 min, and they were studied at day 21–25. Organ baths Animals were deeply anesthetized with pentobarbital sodium (35 mg·kg− 1, i.p.) and exsanguinated. Major airways were dissected and cleaned of connective tissue; four rings were obtained from the middle of the trachea (each ring was submitted to different
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experimental conditions) and hung in a 5 ml organ bath filled with Krebs solution with 1 µM indomethacin, as previously described (Campos-Bedolla et al., 2006). Tissues were stimulated three times with KCl (60 mM), and then temporal course of the responses to 5-HT was evaluated by adding single concentrations of this drug (1, 3.2, 10, 32, 100 and 320 µM) to different tracheal rings. Some of these tissues were preincubated with one of the following drugs during 15 min before addition of a selected concentration of 5-HT (32 µM): tropisetron, methiothepin, ketanserin, WAY-100135, GR 127935, SB 204741, propranolol, L-NAME, ouabain, charybdotoxin and iberiotoxin. Concentrations and descriptions of these drugs are shown in Table 1. None of these drugs modified the basal tone. All responses were expressed as percentage of the third KCl response. We corroborated that the concentration used of ketanserin caused 84% inhibition of the contractile response to a specific 5-HT2A agonist (α-methyl-5-HT, 32 µM) and completely abolished the intracellular Ca2+ peak induced by 100 µM α-methyl-5-HT (data not shown). In order to evaluate the relaxing effect of 5-HT, tracheal rings were precontracted with 10 µM histamine, and then a cumulative concentration–response curve to 5-HT (32, 100 and 320 µM) was done. In some of these tissues, 100 nM charybdotoxin, 32, 100 and 320 nM iberiotoxin or 10 nM ketanserine was added 10 min before histamine administration. In a separate set of experiments, tracheal rings from guinea pigs sensitized to OA were used to evaluate the temporal course of the response to 32 µM 5-HT, which were compared with control (nonsensitized) tissues. Patch clamp recordings Isolated myocytes from guinea pig trachea were obtained as follows. Tracheal airway smooth muscle freed from any residual connective tissue was placed in 5 ml Hanks solution containing 2 mg cysteine and 0.05 U·ml− 1 papaine, and incubated for 10 min at 37 °C. The tissue was washed with Leibovitz's solution to remove the enzyme excess, and then placed in Hanks solution containing 1 mg·ml− 1 collagenase type I and 4 mg·ml− 1 dispase II (neutral protease) during ~20 min at 37 °C. The tissue was gently dispersed by mechanical agitation until detached cells were observed. Enzymatic activity was stopped by adding Leibovitz's solution, the cells were centrifuged at 800 rpm at 20 °C during 5 min and the supernatant was discarded. This last step was repeated once. For myocytes culture, the cell pellet was resuspended in minimum essential medium containing 5% guinea pig serum, 2 mM L-glutamine, 10 U·ml− 1 penicillin, 10 μg·ml− 1 streptomycin and 15 mM glucose, and plated on rounded coverslips coated with sterile rat tail collagen. Cell culture was performed at 37 °C in a 5% CO2 in oxygen during 24–48 h. Airway smooth muscle cells were allowed to settle down in the bottom of a 0.7 ml coverglass submerged in a perfusion chamber. The chamber was perfused by gravity (~ 1.5–2.0 ml·min− 1) with external solution (mM): NaCl 130, KCl 5, CaCl2 1, HEPES 10, glucose 10, MgCl2 0.5, NaHCO3 3, KH2PO4 1.2, and niflumic acid 0.1 (pH 7.4, adjusted
Table 1 Drugs used in the experimental protocols Drug
Description
Concentration
References
Tropisetron Methiothepin Ketanserin WAY-100135 GR 127935 SB 204741 Ouabain Charybdotoxin Iberiotoxin Propranolol L-NAME
5-HT3/5-HT4 antagonist 5-HT1/5-HT2/5-HT5/5-HT6/5-HT7 antagonist 5-HT2A receptor antagonist 5-HT1A receptor antagonist 5-HT1B/5-HT1D receptor antagonist 5-HT2B receptor antagonist Na+/K+-ATPase pump inhibitor Ca2+-activated K+ channel blocker High-conductance Ca2+-activated K+ channel selective blocker β-Adrenoceptor antagonist Inhibitor of NO synthase
1 µM 1 µM 10 nM 100 nM 10 nM 32, 100 nM 10 µM 100 nM 32, 100 or 320 nM 100 nM 10 µM
Pype et al. (1994); Dupont et al. (1996) Gerhardt and Van Heerikhuizen (1997); Kitazawa et al. (2006) Hoyer et al. (2002) Hoyer et al. (2002) Germonpré et al. (1998) Hoyer et al. (2002) Rhoden et al. (2000) Miller et al. (1985) Liu et al. (2007) Campos-Bedolla et al. (2006) Jing et al. (1995)
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with NaOH). Experiments were performed at room temperature (~ 21 °C). The standard whole-cell configuration and an Axopatch 200A amplifier (Axon) were used to record the membrane K+ currents activated by depolarizing voltage steps (i.e., voltage clamp). Patch pipettes were made with 1B200F-6 glass (Word Precision Instruments, Sarasota, FL) using a horizontal micropipette puller (P-87, Sutter Instruments Co, Novato, CA). Pipette resistance ranged from 2 to 4 MΩ. The internal solution was (mM): potassium gluconate 140, NaCl 5, HEPES 5, EGTA 1, ATP disodium 5, GTP sodium 0.1, and leupeptin 0.1 (pH 7.3, adjusted with KOH). Whole-cell currents were filtered at 1–5 kHz using the analogical filter of the amplifier, digitized (Digidata 1200, Axon) at 10 kHz, and stored on a computer for later analysis through special software (pClamp v 8.0, Axon). A series of hyperpolarizing and depolarizing square pulses (from −70 to +40 mV) was applied to the myocytes in 10 mV increments from a holding potential of −60 mV during 500 ms at 1 Hz to observe outward K+ currents. The protocol to evaluate the Ca2+ dependent K+ currents (IK(Ca)) and the effect of 5-HT on these currents was as follows. Once a basal recording of K+ currents were obtained, delayed rectifier K+ channels were blocked by continuously perfusing the cell with 3 mM 4-aminopyridine (4-AP), then the effect of 5-HT was evaluated by adding 32 µM of this drug to the perfusion system, and finally the role of BKCa channels was evaluated by adding 100 nM charybdotoxin or 100 nM iberiotoxin. After each treatment, K+ currents were recorded. To evaluate the role of 5-HT2A receptors, this last protocol was repeated once again but adding 10 nM ketanserin instead of charybdotoxin/iberiotoxin. Intracellular Ca2+ measurements in tracheal myocytes Guinea pig tracheal myocytes were isolated as described above. Cells were loaded with 0.5 µM fura 2/AM in low Ca2+ (0.1 mM) at room temperature (~ 21 °C). After 1 h, cells were allowed to settle down into a heated perfusion chamber with a glass cover in the bottom. This chamber was mounted on an inverted microscope (Diaphot 200, Nikon, Tokyo, Japan) and cells adhered to the glass were continuously perfused at a rate of 2–2.5 ml·min− 1 with Krebs solution (composition in mM: NaCl 118, KCl 4.6, CaCl2 2.0, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11; 37 °C, bubbled with 5% CO2 in oxygen, pH 7.4). Cells loaded with fura 2 were exposed to alternating pulses of 340 and 380 nm excitation light, and emission light was collected at 510 nm using a microphotometer (Photon Technology International, Princeton, NJ). The fluorescence acquisition rate was 0.5 s. Intracellular
Fig. 1. Responses of guinea pig tracheal rings to different concentrations of 5-HT. Biphasic responses (contraction–relaxation) were observed at each 5-HT concentration used. The inset corresponds to a representative recording of the biphasic response induced by 32 µM 5-HT. Symbols represent mean ± SEM.
Fig. 2. Effect of tropisetron (TR) and methiothepin (MET) on the biphasic response induced by 5-HT in guinea pig tracheal rings. Combination of both antagonists almost completely abolished the response to 5-HT. ⁎p b 0.05, and ⁎⁎p b 0.01, as compared with the 5-HT group (one-way ANOVA with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
Ca2+ concentration ([Ca2+]i) was calculated according to the formula by Grynkiewicz et al. (1985). The Kd of fura 2 was assumed to be 386 nM (Kajita and Yamaguchi 1993). The mean 340/380 fluorescence ratios Rmax and Rmin were obtained as previously reported (Carbajal et al., 2005). After corroborating the cell viability through a 10 mM caffeine stimulation, some guinea pig tracheal myocytes were stimulated with 100 µM 5-HT, and this stimuli was repeated once again in the presence of 10 nM ketanserin. In order to indirectly evaluate the activity of the sarcoplasmic reticulum (SR)-ATPase Ca2+ pump, we measured the ability of myocytes to refill their SR Ca2+ stores. Before experiments, viability of the single cells was assessed through stimulation with caffeine in Krebs solution. Then, myocytes were perfused with Ca2+-free solution and 1 min later caffeine (S1) was added during 10 min. The caffeine stimulation in a Ca2+-free medium completely depletes the SR Ca2+ store (Bazan-Perkins et al., 2003). Afterward, cells were washed with Ca2+-free medium to remove caffeine and perfused with Krebs (2.5 mM Ca2+) during 10 min to allow SR Ca2+ refilling. Finally, the stimulation with caffeine was repeated once again (S2) under the same Ca2+-free conditions as before. In these experiments the S2/S1 ratio corresponded to the degree of SR Ca2+ refilling. In some experiments, cells were incubated with 32 µM 5-HT 10 min before S2.
Fig. 3. Decrement of the 5-HT-induced relaxing phase by the 5-HT2A receptor antagonist ketanserin (KT) in guinea pig tracheal rings. ⁎p b 0.05, ⁎⁎p b 0.01 (paired Student's t test). Symbols represent mean ± SEM.
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Fig. 4. Effect of several drugs on the biphasic response induced by 5-HT in guinea pig tracheal rings. WAY-100135 (WAY, a 5-HT1A receptor antagonist), GR 127935 (GR, a 5-HT1B/ 5-HT1D receptor antagonist), SB 204741 (SB, a 5-HT2B receptor antagonist), as well as propranolol (PROP) and L-NAME did not modify the response to 5-HT. Symbols represent mean ±SEM.
Drugs 5-HT hydrochloride, 3-tropanyl-indol-3-carboxylate hydrochloride (tropisetron), methiothepin mesylate, (±)-propranolol hydrochloride, ketanserin tartrate, Nω-nitro- L -arginine methyl ester hydrochloride (L-NAME), ouabain, charybdotoxin, iberiotoxin, histamine dihydrochloride, ovalbumin and caffeine were all purchased from Sigma Chem. Co. (St Louis, MO). (S)-WAY-100135 dihydrochloride, SB 204741 and GR 127935 hydrochloride were purchased from Tocris Cookson Inc. (Ellisville, MO). 4-Aminpyridine was acquired from
Research Chemicals LTD (Ward Hill, MA). Because 5-HT is a lightsensitive chemical compound, all experiments using this drug were performed under dark conditions. Statistical analysis Differences in the response of tracheal rings and [Ca2+]i were evaluated through paired or unpaired Student's t test or one-way ANOVA followed by Dunnett's multiple comparisons test. Patch clamp experiments were evaluated through repeated measures ANOVA
Fig. 5. Effect of charybdotoxin and ouabain (A) or increasing concentrations of iberiotoxin (B) on the relaxing phase induced by 5-HT in guinea pig tracheal rings. The blockade of Ca2+activated K+ channels by charybdotoxin (CTX), and at less extent the blockade of the Na+/K+-ATPase pump by ouabain (OUA), notably averted the relaxation during the response to 5-HT. Likewise, blockade of the high-conductance Ca2+-activated K+ channels by iberiotoxin (IBTX) caused a concentration-dependent diminution of the 5-HT-induced relaxation. ⁎p b 0.05, ⁎⁎p b 0.01 (one-way ANOVA [panel A] or repeated measures ANOVA [panel B] with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
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Fig. 6. Cumulative concentration–response curve to 5-HT in guinea pig tracheal rings precontracted with 10 µM histamine. Effect of charybdotoxin (CTX, panel A), iberiotoxin (IBTX, panel B) and ketanserin (KT, panel C) on the relaxation induced by 5-HT. ⁎p b 0.05, ⁎⁎p b 0.01 (paired Student's t test [panel A, C] and one-way ANOVA [panel B] with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
almost abolished by a combination of both drugs (n = 5). By contrast, incubation with 10 nM ketanserin (n = 6) significantly prevented the relaxing phase of the response to 5-HT (Fig. 3). Finally, as illustrated in Fig. 4, WAY-100135 (n = 6), GR 127935 (n = 6), and SB 204741 (n = 4 each group) did not change the biphasic response induced by 5-HT, nor it was modified by the β-adrenoceptor antagonist propranolol (n = 9) or the NO synthase inhibitor L-NAME (n = 7). Inhibition of the Na+/K+-ATPase pump by ouabain (n = 6) caused a significant reduction of the relaxing phase of the 5-HT response (Fig. 5A). Interestingly, in these experiments a distinctive pattern was constantly observed: after the relaxing response had reached a new steady state, a re-contraction took place as to reach again almost the initial maximal contraction to 5-HT; this re-contraction peaked at approximately 52 ± 4 min after addition of 5-HT. The blockade of Ca2+activated K+ channels by 100 nM charybdotoxin greatly inhibited the 5-HT-induced relaxation (Fig. 5A). A more specific blockade of the high-conductance Ca2+-activated K+ channels with increasing concentrations of iberiotoxin (32, 100 or 320 nM, n = 6 each group) produced a concentration-dependent inhibition of this relaxing phase, which reached statistical significance at 100 and 320 nM (Fig. 5B). We performed some experiments simultaneously using ouabain plus charybdotoxin, but they were useless because this combination of drugs caused a quite irregular and prolonged contraction of tracheal rings, without noticeable changes in [Ca2+]i in single cell experiments (data not shown). At the light of the above-mentioned experiments, we decided to evaluate the relaxing effect of 5-HT by performing a concentration– response curve to 5-HT in tracheas precontracted with histamine (n = 5). These results showed that all 5-HT concentrations used (32, 100 and 320 µM) produced a small relaxation (up to ~27%) (Fig. 6). This relaxing effect was notably abolished by charybdotoxin (n = 5), iberiotoxin (n = 6) or ketanserine (n = 5) (Fig. 6A, B and C, respectively). With the lowest 5-HT concentration (32 µM) a small transient contraction of 21.4 ± 5.7% preceded the relaxation (data not shown). Contrasting with the biphasic nature of the control (nonsensitized) tracheal response to 32 µM 5-HT (n = 7), when tissues were obtained from animals sensitized to OA a more sustained contraction to 5-HT was observed, reaching statistically significant differences from 8 min ahead (p b 0.05 and p b 0.01, n = 6, Fig. 7). In the voltage clamp experiments with single myocytes, outward K+ currents were activated when step depolarizations from −70 to 40 mV were applied from a holding potential of −60 mV (Fig. 8, control group). In order to rule out the voltage-dependent K+ channels (delayed rectifier) all experimental groups received 3 mM 4-AP, which per se caused a significant reduction of control K+ currents (statistics not
followed by Dunnett's multiple comparisons test. Statistical significance was set at two-tailed p b 0.05. Data are expressed in the text and illustrations as mean ± SEM. Results All non-cumulative concentrations of 5-HT induced a biphasic response (contraction followed by relaxation) of tracheal rings (Fig. 1, n = 5–7/group). We choose to use 32 µM in the following experiments because this concentration caused representative biphasic responses. A similar biphasic response to 5-HT was observed in smooth muscle strips free of epithelium and connective tissue (data not shown). The possible role of different 5-HT receptors was evaluated through several antagonists. As can be seen in Fig. 2, the contractile response to 32 µM 5-HT (n = 7) was notably reduced by either tropisetron (n = 5) or methiothepin (n = 5), and such response was
Fig. 7. Effect of sensitization on the 5-HT response in guinea pig tracheal rings. Relaxation phase was greatly diminished in tracheas obtained from animals sensitized to ovalbumin, as compared with control tissues from non-sensitized animals. ⁎p b 0.05, ⁎⁎p b 0.01 (non-paired Student's t test). Symbols represent mean ± SEM.
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shown). Addition of 32 µM 5-HT, in the presence of 4-AP, notably increased K+ currents, which in turn were blocked by 100 nM charybdotoxin (a Ca2+-activated K+ channels blocker) or 100 nM iberiotoxin (a BKCa channels selective blocker), thus corroborating that they corresponded to IK(Ca) currents (Fig. 8A, B). Likewise, in a separate set of experiments using the same protocol, 10 nM ketanserin instead of charybdotoxin completely abolished the IK(Ca) increment induced by 5HT (Fig. 8C). In single airway smooth muscle cells, 5-HT induced a transient Ca2+ peak of 428 ± 142 nM (n = 5). This response was completely abolished by 10 nM ketanserin (Fig. 9A, B, n = 5).
Fig. 9. Example of an intracellular Ca2+ recording in a single myocyte. Panel A illustrates typical recordings where 5-HT induced a transient Ca2+ peak, which was abolished by ketanserin (KT). Panel B shows the statistical analysis demonstrating that KT (n = 5) abolished the effect of 5-HT (n = 5). ⁎p b 0.05 (non-paired Student's t test). Bars represent mean ± SEM.
When the SR-ATPase Ca2+ pump activity was indirectly evaluated through the SR-Ca2+ refilling, control myocytes showed an S2/S1 ratio of 0.68 ± 0.03, (n = 6, Fig. 10), and this ratio was significantly reduced by 32 µM 5-HT (0.4 ± 0.04, n = 5, p b 0.01). Discussion
Fig. 8. Effect of 5-HT on the IK(Ca) currents in a typical relation voltage current. K+ currents were evoked by step depolarization from −70 to 40 mV (control group). 3 mM 4-amynopyridine (4-AP, a delayed rectifier K+ channels blocker) reduced the K+ currents. The addition of 32 µM 5-HT, in the presence of 4-AP, increased the IK(Ca) currents, which were blocked by 100 nM charybdotoxin (CTX, panel A), 100 nM iberiotoxin (IBTX, panel B) or 10 nM ketanserin (KT, panel C). Insets correspond to examples of the original recordings of K+ after each treatment. †p b 0.05, ⁎p b 0.01 (repeated measures ANOVA with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
In the present work we found that BKCa channels, as well as the Na+/K+ATPase pump, play a prominent role in the generation of the 5-HT-induced relaxation. We also determined that these mechanisms are probably triggered by 5-HT2A receptor activation. The contractile effect induced by 5-HT in airway smooth muscle has been widely described in many animal species including humans (Goldie et al., 1982; Lemoine and Kaumann 1986; Doucet et al., 1990; Buckner et al., 1991; Baron et al., 1993; Adner et al., 2002). At least in human bronchi and guinea pig trachea this contraction is followed by a relaxing phase, i.e., a biphasic response is elicited (Goldie et al., 1982; Baumgartner et al., 1990). It has been claimed that this last relaxing phase is mainly observed at high 5-HT concentrations (≥10 µM) (Baumgartner et al., 1990; Ben-Harari et al., 1994). Our results pointed out that such a biphasic response could be observed at all 5-HT concentrations tested (from 1 to 320 µM). Moreover, this relaxing capability of 5-HT was also corroborated in tissues precontracted with histamine. The mechanisms by which 5-HT causes relaxation have not been well documented.
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Fig. 10. Effect of 5-HT on the SR Ca2+ refilling in single myocytes from guinea pig trachea. Panel A shows the experimental protocol to evaluate the SR Ca2+ refilling using caffeine (10 mM). For details see Materials and methods (intracellular Ca2+ measurements in tracheal myocytes). Panel B illustrates typical recordings of 5-HT effect on the SR Ca2+ refilling. Panel C shows the statistical analysis demonstrating that 5-HT (n = 5) reduced the SR Ca2+ refilling (expressed as the S2/S1 ratio), as compared with control group (CTL, n = 6). S1 = first caffeine stimulation, S2 = second caffeine stimulation. ⁎p b 0.01 (nonpaired Student's t test). Bars represent mean ± SEM.
Through the use of several antagonists of different 5-HT receptors we tried to identified potential mechanisms implicated in the response to 5-HT in guinea pig airways, especially those 5-HT receptors already postulated as involved in relaxation. We found that the combination of tropisetron plus methiothepin almost completely abolished the biphasic response to 5-HT, demonstrating that such response was fully dependent on 5-HT receptors activation. The contractile phase has received much attention and it has been
suggested to be mediated at least by activation of 5-HT1A and 5-HT2A receptors in smooth muscle, and modulated by prejunctional 5-HT3 and 5-HT7 receptors (Cazzola and Matera, 2000). The fact that in our experiments ketanserin completely abolished the 5-HT-induced transient Ca2+ peak, but does not avoid the contraction of tracheal rings corroborates that mechanisms other than activation of 5-HT2A receptors are participating in the 5-HT-induced airway smooth muscle contraction (for example, decrease of cAMP by 5-HT1A activation, secondary release of neurotransmitters, blockade of relaxing mechanisms). By contrast, mechanisms involved in the relaxing phase have been scarcely investigated. Baumgartner et al. (1990) were the first who postulated that the 5-HT-induced relaxation was mediated through activation of the 5-HT2 receptor. In our study we corroborated that ketanserin notably reduced this relaxation, confirming the role of 5HT2A receptors in this relaxing response. It is well known that an increased activity of the Na+/K+-ATPase pump induces hiperpolarization of the cell membrane, and thus favors relaxation (Souhrada and Souhrada, 1989). In the vascular smooth muscle, 5-HT has been demonstrated to induce activation of the Na+/K+-ATPase pump, causing a decrement of the vascular tone (Moreland et al., 1985; Fernandez-Alfonso et al., 1992). Rhoden et al. (2000) demonstrated that 5-HT stimulates the Na+/K+ATPase pump in cultured airway smooth muscle cells from guinea pigs via activation of the 5-HT2A receptor, and postulated that under certain circumstances it might give rise to relaxation. Our results indeed support that 5-HT-induced relaxation is partially mediated by activation of the Na+/K+-ATPase pump, and probably mediated via 5-HT2A receptor. Airway smooth muscle possesses a high density of different kinds of K+ channels, which mainly regulate the membrane potential and excitability, thus inducing hyperpolarization and relaxation (Miura et al., 1992; Morley, 1994; Nielsen-Kudsk, 1996; Small et al., 1992; Snetkov et al.,1995,1996; Snetkov and Ward,1999). Major K+ channels in this tissue are: a) voltage-dependent delayed rectifier K+ channels (Kv) (Zhao et al., 2004), which are blocked by 4-AP, b) inward rectifier K+ channels (Kir), which are voltage-regulated (Oonuma et al., 2002) and are blocked by Ba2+, and c) high-conductance Ca2+-activated K+ channels (KCa, Maxi K or BKCa) (Snetkov et al., 1999), which are blocked by charybdotoxin, iberiotoxin and tetraethylamonium. In the present work we found that BKCa channels seem to play a major role during the relaxation induced by 5-HT. This conclusion was drawn from two types of experimental evidences. First, charybdotoxin or iberiotoxin almost completely abolished the 5-HT-induced relaxation in tracheal rings, either in the biphasic response to a single 5-HT concentration or in the concentration–response curve to 5-HT in precontracted tissues. Secondly, with the voltage clamp technique in single myocytes 5-HT increased the IK(Ca) and either charybdotoxin or iberiotoxin avoided such effect. BKCa channels require an increment in the [Ca2+]i in order to be activated (Barrett et al., 1982) and we corroborated that in our experimental conditions 5-HT induced such an [Ca2+]i increase via 5-HT2A receptors. Ketanserin also completely abolished the IK(Ca) increment induced by 5-HT. As far as we know, this is the first report describing that the active relaxation induced by 5-HT is mostly mediated by BKCa channels through 5-HT2A receptors. The potential role of K+ channels other than BKCa remains to be elucidated. Downregulation of the [Ca2+]i through enhanced activity of the SRATPase Ca2+ pump might constitute a potential mechanism inducing smooth muscle relaxation. For example, bronchodilator drugs that increase cAMP concentration enhance SR-ATPase Ca2+ pump activity through inhibition of phospholamban. In its non-phosphorylated state, phospholamban inhibits the SR-ATPase Ca2+ pump, but such inhibitory effect ends when this protein is phosphorylated by cAMPdependent PKA or Ca2+/calmodulin-dependent protein kinase (CaMKII) (Sathish et al., 2008). Thus, we explored the possibility that this mechanism was operating in the 5-HT-induced relaxation. Contrary to
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this hypothesis, we found that 5-HT reduced the activity of the SRATPase Ca2+ pump, which should favor smooth muscle contraction instead of relaxation. Ben-Harari et al. (1991, 1994) by performing experiments in isolated guinea pig tracheas, concluded that a phenomenon of desensitization of 5-HT2 receptors was responsible of the decay of the contraction induced by 5-HT. However, our results showed that ouabain, charybdotoxin or iberiotoxin effectively diminished or even abolished such relaxation, and 5-HT added to precontracted tracheas induced relaxation. These findings pointed out that an active relaxation was occurring, and thus mechanisms other than desensitization could also be involved in the relaxation phase. Involvement of another type of 5-HT receptor, namely the 5-HT1 receptor, in the relaxation induced by 5-HT has been proposed by D'Agostino et al. (1996). However, in our study we found that WAY100135 and GR 127935, two specific antagonists of 5-HT1A and 5-HT1B/ 5-HT1D receptors, respectively, were unable to modify the response to 5-HT. Interestingly, higher concentrations of WAY-100135 (10 µM) partially inhibited the relaxation phase of the 5-HT response. Nevertheless, we also found that at this high concentration WAY-100135 significantly reduced the intracellular Ca2+ peak induced by 5-HT in single airway smooth muscle cells from guinea pig (a response exclusively mediated by 5-HT2A receptors) (data not shown), which pointed out that at this concentration this compound lost its selectivity and also antagonizes 5-HT2A receptors. Two other potential relaxing mechanisms, β-adrenoceptor activation and nitric oxide production, were also evaluated in our study. Both mechanisms were discarded inasmuch as propranolol and LNAME did not change the biphasic response to 5-HT. The lack of effect of propranolol is in contrast with a previous study of our group with a closely-related drug, α-methyl-5-HT (a 5-HT2 agonist) (CamposBedolla et al., 2006). Like 5-HT, this drug also caused a biphasic response in guinea pig tracheas, but we found that β2-adrenoceptors were involved in the relaxation phase. As we commented above, pulmonary function in asthmatic children was improved by the administration of tianeptine, a drug that lowers plasma 5-HT by enhancing the re-uptake of 5-HT (Lechin et al., 1998), suggesting a role of 5-HT in the pathophysiology of asthma. Interestingly enough, our in vitro experiments showed that the biphasic nature of the response to 5-HT was notably changed to a more sustained contraction in sensitized tissues. This finding might implicate that a relaxation mechanism induced by 5-HT is impaired by sensitization. To what extent the sensitization procedure caused an abnormality of the high-conductance Ca2+-activated K+ channels is unknown. This and other potential mechanisms triggered by the sensitization procedure deserve further investigation. We concluded that during the relaxation induced by 5-HT two major mechanisms seem to be involved: the stimulation of the Na+/K+ATPase pump, and an increased activity of the BKCa channels, both of them probably mediated via 5-HT2A receptors. Acknowledgments This work is part of the PhD degree of Patricia Campos-Bedolla, and we thank Dr. Israel Grijalva for his support in the development of this research. This study was supported by grants from DGAPA-UNAM (IN202107) to Dr. Luis M. Montaño, and from IMSS (2006-1A-I-077) to MSc Patricia Campos-Bedolla. References Adner, M., Rose, A.C., Zhang, Y., Sward, K., Benson, M., Uddman, R., Shankley, N.P., Cardell, L.O., 2002. An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNF-α up-regulates 5HT2A-mediated contraction. British Journal of Pharmacology 137 (7), 971–982. Baron, C.B., Pompeo, J., Blackman, D., Coburn, R.F., 1993. Common phosphatidylinositol 4,5-bisphosphate pools are involved in carbachol and serotonin activation of
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Airway smooth muscle relaxation induced by 5-HT2A receptors: Role of Na+/K+-ATPase pump and Ca2+-activated K+ channels Patricia Campos-Bedolla a, Mario H. Vargas b, Patricia Segura b, Verónica Carbajal b, Eduardo Calixto c, Alejandra Figueroa e, Edgar Flores-Soto e, Carlos Barajas-López d, Nicandro Mendoza-Patiño e, Luis M. Montaño e,⁎ a
Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México DF, Mexico Departamento de Investigación en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, México DF, Mexico c Departamento de Neurobiología, División de Investigación en Neurociencias, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, México DF, Mexico d División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, SLP, Mexico e Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, México DF, Mexico b
a r t i c l e
i n f o
Article history: Received 14 February 2008 Accepted 17 July 2008 Keywords: Airway smooth muscle Relaxation 5-HT2A receptors BKCa channels Na+/K+-ATPase pump
a b s t r a c t Aims: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. Main methods: Using organ baths, patch clamp, and intracellular Ca2+ measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. Key findings: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 μM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 μM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT1A, 5-HT1B/5-HT1D and 5-HT2B receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 μM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K+ currents (IK(Ca)), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. Significance: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na+/K+-ATPase pump, and increasing activity of the high-conductance Ca2+-activated K+ channels, probably via 5-HT2A receptors. © 2008 Elsevier Inc. All rights reserved.
Introduction 5-Hydroxytryptamine (5-HT or serotonin) is an important neurotransmitter of the central nervous system and the digestive tract, and has a major role in some conditions such as migraine and inflammatory bowel disease. Serotonergic fibers have not been described in the respiratory system, but in different mammalian species, including humans, there are non-neuronal sources of 5-HT such as mast cells and neuroendocrine cells (Joos et al., 1997; Lauweryns et al., 1974; Fu et al., 2002). Moreover, in pulmonary tissue, 5-HT levels are directly proportional to their plasmatic ⁎ Corresponding author. Departamento de Farmacología, Edificio de Investigación, sexto piso, laboratorio 3, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510, México DF, Mexico. Tel./fax: +55 56665868. E-mail address: [email protected] (L.M. Montaño). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.07.006
concentrations, being the platelets as its main source (Cazzola and Matera 2000). The role of 5-HT in asthma has been controversial, but there are some clues indicating its potential involvement in this disease. Plasma concentration of free 5-HT notably increases during an asthmatic exacerbation and this increment is related to asthma severity (Lechin et al., 1996). In addition, it has been reported that tianeptine (a drug that lowers plasma 5-HT by enhancing the 5-HT re-uptake) improves pulmonary function in asthmatic children (Lechin et al., 1998). Therefore, it is important to better understand the physiologic effect of 5-HT on the airway smooth muscle. 5-HT induces a direct sustained contraction of airway smooth muscle from bovine, dog, equine or mouse (Goldie et al., 1982; Lemoine and Kaumann 1986; Doucett et al., 1990; Buckner et al., 1991; Baron et al., 1993; Adner et al., 2002), but in other preparations such as guinea pig trachea or human bronchi the responses elicited by 5-HT
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are biphasic (contraction followed by relaxation) in nature (Goldie et al., 1982; Baumgartner et al., 1990; Ben-Harari et al., 1994). The relaxing phase of this biphasic response has been proposed to occur only at 5-HT concentrations ≥10 µM (Baumgartner et al., 1990), and it has been postulated that 5-HT2 receptor is the main serotonergic receptor involved in this effect. Further characterization of the mechanisms involved in the 5-HT-induced relaxation has been scarcely investigated. Baumgartner et al. (1990) described that the relaxation caused by high 5-HT concentrations in guinea pig tracheal strips was coincident with a decrease of IP3 production, and they postulated that an increase in the cAMP might be involved. By evaluating the ouabain-sensitive 86Rb+ uptake in cultured guinea pig tracheal smooth muscle cells, Rhoden et al. (2000) found that 5-HT stimulated the activity of Na+/K+-ATPase via 5-HT2A receptors, but these authors did not explore the physiological consequences of such stimulation. Finally, Ben-Harari et al. (1994) postulated that the relaxation phase induced by a single concentration of 5-HT was related to a receptor-dependent desensitization. The present work was aimed to investigate the potential role of several relaxing mechanisms triggered by 5-HT in guinea pig airway smooth muscle, including the role of Na+/K+-ATPase and highconductance Ca2+-activated K+ (BKCa) channels. Materials and methods Animals Male Hartley guinea pigs (500–600 g) bred in conventional conditions in our institutional animal facilities (filtered conditioned air, 21 ± 1 °C, 50–70% humidity, sterilized bed) and fed with Harlan® pellets and sterilized water were used. The protocol was approved by the Scientific and Bioethics Committees of the Instituto Nacional de Enfermedades Respiratorias. The experiments were conducted in accordance with the published Guiding Principles in the Care and Use of Animals, approved by the American Physiological Society. Sensitization procedure and antigenic challenge Guinea pigs were sensitized at day 0 by intraperitoneal administration of 60 mg ovalbumin (OA) and 1 mg Al(OH)3 in 0.5 ml of saline (0.9% NaCl). At day 8, the animals were nebulized with 3 mg·ml− 1 OA in saline for 5 min, delivered by a ultrasonic nebulizer (model US-1, Puritan Bennett, Carlsbad, CA). Guinea pigs were nebulized again on day 15 with 0.5 mg·ml− 1 OA in saline for 1 min, and they were studied at day 21–25. Organ baths Animals were deeply anesthetized with pentobarbital sodium (35 mg·kg− 1, i.p.) and exsanguinated. Major airways were dissected and cleaned of connective tissue; four rings were obtained from the middle of the trachea (each ring was submitted to different
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experimental conditions) and hung in a 5 ml organ bath filled with Krebs solution with 1 µM indomethacin, as previously described (Campos-Bedolla et al., 2006). Tissues were stimulated three times with KCl (60 mM), and then temporal course of the responses to 5-HT was evaluated by adding single concentrations of this drug (1, 3.2, 10, 32, 100 and 320 µM) to different tracheal rings. Some of these tissues were preincubated with one of the following drugs during 15 min before addition of a selected concentration of 5-HT (32 µM): tropisetron, methiothepin, ketanserin, WAY-100135, GR 127935, SB 204741, propranolol, L-NAME, ouabain, charybdotoxin and iberiotoxin. Concentrations and descriptions of these drugs are shown in Table 1. None of these drugs modified the basal tone. All responses were expressed as percentage of the third KCl response. We corroborated that the concentration used of ketanserin caused 84% inhibition of the contractile response to a specific 5-HT2A agonist (α-methyl-5-HT, 32 µM) and completely abolished the intracellular Ca2+ peak induced by 100 µM α-methyl-5-HT (data not shown). In order to evaluate the relaxing effect of 5-HT, tracheal rings were precontracted with 10 µM histamine, and then a cumulative concentration–response curve to 5-HT (32, 100 and 320 µM) was done. In some of these tissues, 100 nM charybdotoxin, 32, 100 and 320 nM iberiotoxin or 10 nM ketanserine was added 10 min before histamine administration. In a separate set of experiments, tracheal rings from guinea pigs sensitized to OA were used to evaluate the temporal course of the response to 32 µM 5-HT, which were compared with control (nonsensitized) tissues. Patch clamp recordings Isolated myocytes from guinea pig trachea were obtained as follows. Tracheal airway smooth muscle freed from any residual connective tissue was placed in 5 ml Hanks solution containing 2 mg cysteine and 0.05 U·ml− 1 papaine, and incubated for 10 min at 37 °C. The tissue was washed with Leibovitz's solution to remove the enzyme excess, and then placed in Hanks solution containing 1 mg·ml− 1 collagenase type I and 4 mg·ml− 1 dispase II (neutral protease) during ~20 min at 37 °C. The tissue was gently dispersed by mechanical agitation until detached cells were observed. Enzymatic activity was stopped by adding Leibovitz's solution, the cells were centrifuged at 800 rpm at 20 °C during 5 min and the supernatant was discarded. This last step was repeated once. For myocytes culture, the cell pellet was resuspended in minimum essential medium containing 5% guinea pig serum, 2 mM L-glutamine, 10 U·ml− 1 penicillin, 10 μg·ml− 1 streptomycin and 15 mM glucose, and plated on rounded coverslips coated with sterile rat tail collagen. Cell culture was performed at 37 °C in a 5% CO2 in oxygen during 24–48 h. Airway smooth muscle cells were allowed to settle down in the bottom of a 0.7 ml coverglass submerged in a perfusion chamber. The chamber was perfused by gravity (~ 1.5–2.0 ml·min− 1) with external solution (mM): NaCl 130, KCl 5, CaCl2 1, HEPES 10, glucose 10, MgCl2 0.5, NaHCO3 3, KH2PO4 1.2, and niflumic acid 0.1 (pH 7.4, adjusted
Table 1 Drugs used in the experimental protocols Drug
Description
Concentration
References
Tropisetron Methiothepin Ketanserin WAY-100135 GR 127935 SB 204741 Ouabain Charybdotoxin Iberiotoxin Propranolol L-NAME
5-HT3/5-HT4 antagonist 5-HT1/5-HT2/5-HT5/5-HT6/5-HT7 antagonist 5-HT2A receptor antagonist 5-HT1A receptor antagonist 5-HT1B/5-HT1D receptor antagonist 5-HT2B receptor antagonist Na+/K+-ATPase pump inhibitor Ca2+-activated K+ channel blocker High-conductance Ca2+-activated K+ channel selective blocker β-Adrenoceptor antagonist Inhibitor of NO synthase
1 µM 1 µM 10 nM 100 nM 10 nM 32, 100 nM 10 µM 100 nM 32, 100 or 320 nM 100 nM 10 µM
Pype et al. (1994); Dupont et al. (1996) Gerhardt and Van Heerikhuizen (1997); Kitazawa et al. (2006) Hoyer et al. (2002) Hoyer et al. (2002) Germonpré et al. (1998) Hoyer et al. (2002) Rhoden et al. (2000) Miller et al. (1985) Liu et al. (2007) Campos-Bedolla et al. (2006) Jing et al. (1995)
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with NaOH). Experiments were performed at room temperature (~ 21 °C). The standard whole-cell configuration and an Axopatch 200A amplifier (Axon) were used to record the membrane K+ currents activated by depolarizing voltage steps (i.e., voltage clamp). Patch pipettes were made with 1B200F-6 glass (Word Precision Instruments, Sarasota, FL) using a horizontal micropipette puller (P-87, Sutter Instruments Co, Novato, CA). Pipette resistance ranged from 2 to 4 MΩ. The internal solution was (mM): potassium gluconate 140, NaCl 5, HEPES 5, EGTA 1, ATP disodium 5, GTP sodium 0.1, and leupeptin 0.1 (pH 7.3, adjusted with KOH). Whole-cell currents were filtered at 1–5 kHz using the analogical filter of the amplifier, digitized (Digidata 1200, Axon) at 10 kHz, and stored on a computer for later analysis through special software (pClamp v 8.0, Axon). A series of hyperpolarizing and depolarizing square pulses (from −70 to +40 mV) was applied to the myocytes in 10 mV increments from a holding potential of −60 mV during 500 ms at 1 Hz to observe outward K+ currents. The protocol to evaluate the Ca2+ dependent K+ currents (IK(Ca)) and the effect of 5-HT on these currents was as follows. Once a basal recording of K+ currents were obtained, delayed rectifier K+ channels were blocked by continuously perfusing the cell with 3 mM 4-aminopyridine (4-AP), then the effect of 5-HT was evaluated by adding 32 µM of this drug to the perfusion system, and finally the role of BKCa channels was evaluated by adding 100 nM charybdotoxin or 100 nM iberiotoxin. After each treatment, K+ currents were recorded. To evaluate the role of 5-HT2A receptors, this last protocol was repeated once again but adding 10 nM ketanserin instead of charybdotoxin/iberiotoxin. Intracellular Ca2+ measurements in tracheal myocytes Guinea pig tracheal myocytes were isolated as described above. Cells were loaded with 0.5 µM fura 2/AM in low Ca2+ (0.1 mM) at room temperature (~ 21 °C). After 1 h, cells were allowed to settle down into a heated perfusion chamber with a glass cover in the bottom. This chamber was mounted on an inverted microscope (Diaphot 200, Nikon, Tokyo, Japan) and cells adhered to the glass were continuously perfused at a rate of 2–2.5 ml·min− 1 with Krebs solution (composition in mM: NaCl 118, KCl 4.6, CaCl2 2.0, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11; 37 °C, bubbled with 5% CO2 in oxygen, pH 7.4). Cells loaded with fura 2 were exposed to alternating pulses of 340 and 380 nm excitation light, and emission light was collected at 510 nm using a microphotometer (Photon Technology International, Princeton, NJ). The fluorescence acquisition rate was 0.5 s. Intracellular
Fig. 1. Responses of guinea pig tracheal rings to different concentrations of 5-HT. Biphasic responses (contraction–relaxation) were observed at each 5-HT concentration used. The inset corresponds to a representative recording of the biphasic response induced by 32 µM 5-HT. Symbols represent mean ± SEM.
Fig. 2. Effect of tropisetron (TR) and methiothepin (MET) on the biphasic response induced by 5-HT in guinea pig tracheal rings. Combination of both antagonists almost completely abolished the response to 5-HT. ⁎p b 0.05, and ⁎⁎p b 0.01, as compared with the 5-HT group (one-way ANOVA with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
Ca2+ concentration ([Ca2+]i) was calculated according to the formula by Grynkiewicz et al. (1985). The Kd of fura 2 was assumed to be 386 nM (Kajita and Yamaguchi 1993). The mean 340/380 fluorescence ratios Rmax and Rmin were obtained as previously reported (Carbajal et al., 2005). After corroborating the cell viability through a 10 mM caffeine stimulation, some guinea pig tracheal myocytes were stimulated with 100 µM 5-HT, and this stimuli was repeated once again in the presence of 10 nM ketanserin. In order to indirectly evaluate the activity of the sarcoplasmic reticulum (SR)-ATPase Ca2+ pump, we measured the ability of myocytes to refill their SR Ca2+ stores. Before experiments, viability of the single cells was assessed through stimulation with caffeine in Krebs solution. Then, myocytes were perfused with Ca2+-free solution and 1 min later caffeine (S1) was added during 10 min. The caffeine stimulation in a Ca2+-free medium completely depletes the SR Ca2+ store (Bazan-Perkins et al., 2003). Afterward, cells were washed with Ca2+-free medium to remove caffeine and perfused with Krebs (2.5 mM Ca2+) during 10 min to allow SR Ca2+ refilling. Finally, the stimulation with caffeine was repeated once again (S2) under the same Ca2+-free conditions as before. In these experiments the S2/S1 ratio corresponded to the degree of SR Ca2+ refilling. In some experiments, cells were incubated with 32 µM 5-HT 10 min before S2.
Fig. 3. Decrement of the 5-HT-induced relaxing phase by the 5-HT2A receptor antagonist ketanserin (KT) in guinea pig tracheal rings. ⁎p b 0.05, ⁎⁎p b 0.01 (paired Student's t test). Symbols represent mean ± SEM.
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Fig. 4. Effect of several drugs on the biphasic response induced by 5-HT in guinea pig tracheal rings. WAY-100135 (WAY, a 5-HT1A receptor antagonist), GR 127935 (GR, a 5-HT1B/ 5-HT1D receptor antagonist), SB 204741 (SB, a 5-HT2B receptor antagonist), as well as propranolol (PROP) and L-NAME did not modify the response to 5-HT. Symbols represent mean ±SEM.
Drugs 5-HT hydrochloride, 3-tropanyl-indol-3-carboxylate hydrochloride (tropisetron), methiothepin mesylate, (±)-propranolol hydrochloride, ketanserin tartrate, Nω-nitro- L -arginine methyl ester hydrochloride (L-NAME), ouabain, charybdotoxin, iberiotoxin, histamine dihydrochloride, ovalbumin and caffeine were all purchased from Sigma Chem. Co. (St Louis, MO). (S)-WAY-100135 dihydrochloride, SB 204741 and GR 127935 hydrochloride were purchased from Tocris Cookson Inc. (Ellisville, MO). 4-Aminpyridine was acquired from
Research Chemicals LTD (Ward Hill, MA). Because 5-HT is a lightsensitive chemical compound, all experiments using this drug were performed under dark conditions. Statistical analysis Differences in the response of tracheal rings and [Ca2+]i were evaluated through paired or unpaired Student's t test or one-way ANOVA followed by Dunnett's multiple comparisons test. Patch clamp experiments were evaluated through repeated measures ANOVA
Fig. 5. Effect of charybdotoxin and ouabain (A) or increasing concentrations of iberiotoxin (B) on the relaxing phase induced by 5-HT in guinea pig tracheal rings. The blockade of Ca2+activated K+ channels by charybdotoxin (CTX), and at less extent the blockade of the Na+/K+-ATPase pump by ouabain (OUA), notably averted the relaxation during the response to 5-HT. Likewise, blockade of the high-conductance Ca2+-activated K+ channels by iberiotoxin (IBTX) caused a concentration-dependent diminution of the 5-HT-induced relaxation. ⁎p b 0.05, ⁎⁎p b 0.01 (one-way ANOVA [panel A] or repeated measures ANOVA [panel B] with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
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Fig. 6. Cumulative concentration–response curve to 5-HT in guinea pig tracheal rings precontracted with 10 µM histamine. Effect of charybdotoxin (CTX, panel A), iberiotoxin (IBTX, panel B) and ketanserin (KT, panel C) on the relaxation induced by 5-HT. ⁎p b 0.05, ⁎⁎p b 0.01 (paired Student's t test [panel A, C] and one-way ANOVA [panel B] with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
almost abolished by a combination of both drugs (n = 5). By contrast, incubation with 10 nM ketanserin (n = 6) significantly prevented the relaxing phase of the response to 5-HT (Fig. 3). Finally, as illustrated in Fig. 4, WAY-100135 (n = 6), GR 127935 (n = 6), and SB 204741 (n = 4 each group) did not change the biphasic response induced by 5-HT, nor it was modified by the β-adrenoceptor antagonist propranolol (n = 9) or the NO synthase inhibitor L-NAME (n = 7). Inhibition of the Na+/K+-ATPase pump by ouabain (n = 6) caused a significant reduction of the relaxing phase of the 5-HT response (Fig. 5A). Interestingly, in these experiments a distinctive pattern was constantly observed: after the relaxing response had reached a new steady state, a re-contraction took place as to reach again almost the initial maximal contraction to 5-HT; this re-contraction peaked at approximately 52 ± 4 min after addition of 5-HT. The blockade of Ca2+activated K+ channels by 100 nM charybdotoxin greatly inhibited the 5-HT-induced relaxation (Fig. 5A). A more specific blockade of the high-conductance Ca2+-activated K+ channels with increasing concentrations of iberiotoxin (32, 100 or 320 nM, n = 6 each group) produced a concentration-dependent inhibition of this relaxing phase, which reached statistical significance at 100 and 320 nM (Fig. 5B). We performed some experiments simultaneously using ouabain plus charybdotoxin, but they were useless because this combination of drugs caused a quite irregular and prolonged contraction of tracheal rings, without noticeable changes in [Ca2+]i in single cell experiments (data not shown). At the light of the above-mentioned experiments, we decided to evaluate the relaxing effect of 5-HT by performing a concentration– response curve to 5-HT in tracheas precontracted with histamine (n = 5). These results showed that all 5-HT concentrations used (32, 100 and 320 µM) produced a small relaxation (up to ~27%) (Fig. 6). This relaxing effect was notably abolished by charybdotoxin (n = 5), iberiotoxin (n = 6) or ketanserine (n = 5) (Fig. 6A, B and C, respectively). With the lowest 5-HT concentration (32 µM) a small transient contraction of 21.4 ± 5.7% preceded the relaxation (data not shown). Contrasting with the biphasic nature of the control (nonsensitized) tracheal response to 32 µM 5-HT (n = 7), when tissues were obtained from animals sensitized to OA a more sustained contraction to 5-HT was observed, reaching statistically significant differences from 8 min ahead (p b 0.05 and p b 0.01, n = 6, Fig. 7). In the voltage clamp experiments with single myocytes, outward K+ currents were activated when step depolarizations from −70 to 40 mV were applied from a holding potential of −60 mV (Fig. 8, control group). In order to rule out the voltage-dependent K+ channels (delayed rectifier) all experimental groups received 3 mM 4-AP, which per se caused a significant reduction of control K+ currents (statistics not
followed by Dunnett's multiple comparisons test. Statistical significance was set at two-tailed p b 0.05. Data are expressed in the text and illustrations as mean ± SEM. Results All non-cumulative concentrations of 5-HT induced a biphasic response (contraction followed by relaxation) of tracheal rings (Fig. 1, n = 5–7/group). We choose to use 32 µM in the following experiments because this concentration caused representative biphasic responses. A similar biphasic response to 5-HT was observed in smooth muscle strips free of epithelium and connective tissue (data not shown). The possible role of different 5-HT receptors was evaluated through several antagonists. As can be seen in Fig. 2, the contractile response to 32 µM 5-HT (n = 7) was notably reduced by either tropisetron (n = 5) or methiothepin (n = 5), and such response was
Fig. 7. Effect of sensitization on the 5-HT response in guinea pig tracheal rings. Relaxation phase was greatly diminished in tracheas obtained from animals sensitized to ovalbumin, as compared with control tissues from non-sensitized animals. ⁎p b 0.05, ⁎⁎p b 0.01 (non-paired Student's t test). Symbols represent mean ± SEM.
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shown). Addition of 32 µM 5-HT, in the presence of 4-AP, notably increased K+ currents, which in turn were blocked by 100 nM charybdotoxin (a Ca2+-activated K+ channels blocker) or 100 nM iberiotoxin (a BKCa channels selective blocker), thus corroborating that they corresponded to IK(Ca) currents (Fig. 8A, B). Likewise, in a separate set of experiments using the same protocol, 10 nM ketanserin instead of charybdotoxin completely abolished the IK(Ca) increment induced by 5HT (Fig. 8C). In single airway smooth muscle cells, 5-HT induced a transient Ca2+ peak of 428 ± 142 nM (n = 5). This response was completely abolished by 10 nM ketanserin (Fig. 9A, B, n = 5).
Fig. 9. Example of an intracellular Ca2+ recording in a single myocyte. Panel A illustrates typical recordings where 5-HT induced a transient Ca2+ peak, which was abolished by ketanserin (KT). Panel B shows the statistical analysis demonstrating that KT (n = 5) abolished the effect of 5-HT (n = 5). ⁎p b 0.05 (non-paired Student's t test). Bars represent mean ± SEM.
When the SR-ATPase Ca2+ pump activity was indirectly evaluated through the SR-Ca2+ refilling, control myocytes showed an S2/S1 ratio of 0.68 ± 0.03, (n = 6, Fig. 10), and this ratio was significantly reduced by 32 µM 5-HT (0.4 ± 0.04, n = 5, p b 0.01). Discussion
Fig. 8. Effect of 5-HT on the IK(Ca) currents in a typical relation voltage current. K+ currents were evoked by step depolarization from −70 to 40 mV (control group). 3 mM 4-amynopyridine (4-AP, a delayed rectifier K+ channels blocker) reduced the K+ currents. The addition of 32 µM 5-HT, in the presence of 4-AP, increased the IK(Ca) currents, which were blocked by 100 nM charybdotoxin (CTX, panel A), 100 nM iberiotoxin (IBTX, panel B) or 10 nM ketanserin (KT, panel C). Insets correspond to examples of the original recordings of K+ after each treatment. †p b 0.05, ⁎p b 0.01 (repeated measures ANOVA with Dunnett's multiple comparisons test). Symbols represent mean ± SEM.
In the present work we found that BKCa channels, as well as the Na+/K+ATPase pump, play a prominent role in the generation of the 5-HT-induced relaxation. We also determined that these mechanisms are probably triggered by 5-HT2A receptor activation. The contractile effect induced by 5-HT in airway smooth muscle has been widely described in many animal species including humans (Goldie et al., 1982; Lemoine and Kaumann 1986; Doucet et al., 1990; Buckner et al., 1991; Baron et al., 1993; Adner et al., 2002). At least in human bronchi and guinea pig trachea this contraction is followed by a relaxing phase, i.e., a biphasic response is elicited (Goldie et al., 1982; Baumgartner et al., 1990). It has been claimed that this last relaxing phase is mainly observed at high 5-HT concentrations (≥10 µM) (Baumgartner et al., 1990; Ben-Harari et al., 1994). Our results pointed out that such a biphasic response could be observed at all 5-HT concentrations tested (from 1 to 320 µM). Moreover, this relaxing capability of 5-HT was also corroborated in tissues precontracted with histamine. The mechanisms by which 5-HT causes relaxation have not been well documented.
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Fig. 10. Effect of 5-HT on the SR Ca2+ refilling in single myocytes from guinea pig trachea. Panel A shows the experimental protocol to evaluate the SR Ca2+ refilling using caffeine (10 mM). For details see Materials and methods (intracellular Ca2+ measurements in tracheal myocytes). Panel B illustrates typical recordings of 5-HT effect on the SR Ca2+ refilling. Panel C shows the statistical analysis demonstrating that 5-HT (n = 5) reduced the SR Ca2+ refilling (expressed as the S2/S1 ratio), as compared with control group (CTL, n = 6). S1 = first caffeine stimulation, S2 = second caffeine stimulation. ⁎p b 0.01 (nonpaired Student's t test). Bars represent mean ± SEM.
Through the use of several antagonists of different 5-HT receptors we tried to identified potential mechanisms implicated in the response to 5-HT in guinea pig airways, especially those 5-HT receptors already postulated as involved in relaxation. We found that the combination of tropisetron plus methiothepin almost completely abolished the biphasic response to 5-HT, demonstrating that such response was fully dependent on 5-HT receptors activation. The contractile phase has received much attention and it has been
suggested to be mediated at least by activation of 5-HT1A and 5-HT2A receptors in smooth muscle, and modulated by prejunctional 5-HT3 and 5-HT7 receptors (Cazzola and Matera, 2000). The fact that in our experiments ketanserin completely abolished the 5-HT-induced transient Ca2+ peak, but does not avoid the contraction of tracheal rings corroborates that mechanisms other than activation of 5-HT2A receptors are participating in the 5-HT-induced airway smooth muscle contraction (for example, decrease of cAMP by 5-HT1A activation, secondary release of neurotransmitters, blockade of relaxing mechanisms). By contrast, mechanisms involved in the relaxing phase have been scarcely investigated. Baumgartner et al. (1990) were the first who postulated that the 5-HT-induced relaxation was mediated through activation of the 5-HT2 receptor. In our study we corroborated that ketanserin notably reduced this relaxation, confirming the role of 5HT2A receptors in this relaxing response. It is well known that an increased activity of the Na+/K+-ATPase pump induces hiperpolarization of the cell membrane, and thus favors relaxation (Souhrada and Souhrada, 1989). In the vascular smooth muscle, 5-HT has been demonstrated to induce activation of the Na+/K+-ATPase pump, causing a decrement of the vascular tone (Moreland et al., 1985; Fernandez-Alfonso et al., 1992). Rhoden et al. (2000) demonstrated that 5-HT stimulates the Na+/K+ATPase pump in cultured airway smooth muscle cells from guinea pigs via activation of the 5-HT2A receptor, and postulated that under certain circumstances it might give rise to relaxation. Our results indeed support that 5-HT-induced relaxation is partially mediated by activation of the Na+/K+-ATPase pump, and probably mediated via 5-HT2A receptor. Airway smooth muscle possesses a high density of different kinds of K+ channels, which mainly regulate the membrane potential and excitability, thus inducing hyperpolarization and relaxation (Miura et al., 1992; Morley, 1994; Nielsen-Kudsk, 1996; Small et al., 1992; Snetkov et al.,1995,1996; Snetkov and Ward,1999). Major K+ channels in this tissue are: a) voltage-dependent delayed rectifier K+ channels (Kv) (Zhao et al., 2004), which are blocked by 4-AP, b) inward rectifier K+ channels (Kir), which are voltage-regulated (Oonuma et al., 2002) and are blocked by Ba2+, and c) high-conductance Ca2+-activated K+ channels (KCa, Maxi K or BKCa) (Snetkov et al., 1999), which are blocked by charybdotoxin, iberiotoxin and tetraethylamonium. In the present work we found that BKCa channels seem to play a major role during the relaxation induced by 5-HT. This conclusion was drawn from two types of experimental evidences. First, charybdotoxin or iberiotoxin almost completely abolished the 5-HT-induced relaxation in tracheal rings, either in the biphasic response to a single 5-HT concentration or in the concentration–response curve to 5-HT in precontracted tissues. Secondly, with the voltage clamp technique in single myocytes 5-HT increased the IK(Ca) and either charybdotoxin or iberiotoxin avoided such effect. BKCa channels require an increment in the [Ca2+]i in order to be activated (Barrett et al., 1982) and we corroborated that in our experimental conditions 5-HT induced such an [Ca2+]i increase via 5-HT2A receptors. Ketanserin also completely abolished the IK(Ca) increment induced by 5-HT. As far as we know, this is the first report describing that the active relaxation induced by 5-HT is mostly mediated by BKCa channels through 5-HT2A receptors. The potential role of K+ channels other than BKCa remains to be elucidated. Downregulation of the [Ca2+]i through enhanced activity of the SRATPase Ca2+ pump might constitute a potential mechanism inducing smooth muscle relaxation. For example, bronchodilator drugs that increase cAMP concentration enhance SR-ATPase Ca2+ pump activity through inhibition of phospholamban. In its non-phosphorylated state, phospholamban inhibits the SR-ATPase Ca2+ pump, but such inhibitory effect ends when this protein is phosphorylated by cAMPdependent PKA or Ca2+/calmodulin-dependent protein kinase (CaMKII) (Sathish et al., 2008). Thus, we explored the possibility that this mechanism was operating in the 5-HT-induced relaxation. Contrary to
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this hypothesis, we found that 5-HT reduced the activity of the SRATPase Ca2+ pump, which should favor smooth muscle contraction instead of relaxation. Ben-Harari et al. (1991, 1994) by performing experiments in isolated guinea pig tracheas, concluded that a phenomenon of desensitization of 5-HT2 receptors was responsible of the decay of the contraction induced by 5-HT. However, our results showed that ouabain, charybdotoxin or iberiotoxin effectively diminished or even abolished such relaxation, and 5-HT added to precontracted tracheas induced relaxation. These findings pointed out that an active relaxation was occurring, and thus mechanisms other than desensitization could also be involved in the relaxation phase. Involvement of another type of 5-HT receptor, namely the 5-HT1 receptor, in the relaxation induced by 5-HT has been proposed by D'Agostino et al. (1996). However, in our study we found that WAY100135 and GR 127935, two specific antagonists of 5-HT1A and 5-HT1B/ 5-HT1D receptors, respectively, were unable to modify the response to 5-HT. Interestingly, higher concentrations of WAY-100135 (10 µM) partially inhibited the relaxation phase of the 5-HT response. Nevertheless, we also found that at this high concentration WAY-100135 significantly reduced the intracellular Ca2+ peak induced by 5-HT in single airway smooth muscle cells from guinea pig (a response exclusively mediated by 5-HT2A receptors) (data not shown), which pointed out that at this concentration this compound lost its selectivity and also antagonizes 5-HT2A receptors. Two other potential relaxing mechanisms, β-adrenoceptor activation and nitric oxide production, were also evaluated in our study. Both mechanisms were discarded inasmuch as propranolol and LNAME did not change the biphasic response to 5-HT. The lack of effect of propranolol is in contrast with a previous study of our group with a closely-related drug, α-methyl-5-HT (a 5-HT2 agonist) (CamposBedolla et al., 2006). Like 5-HT, this drug also caused a biphasic response in guinea pig tracheas, but we found that β2-adrenoceptors were involved in the relaxation phase. As we commented above, pulmonary function in asthmatic children was improved by the administration of tianeptine, a drug that lowers plasma 5-HT by enhancing the re-uptake of 5-HT (Lechin et al., 1998), suggesting a role of 5-HT in the pathophysiology of asthma. Interestingly enough, our in vitro experiments showed that the biphasic nature of the response to 5-HT was notably changed to a more sustained contraction in sensitized tissues. This finding might implicate that a relaxation mechanism induced by 5-HT is impaired by sensitization. To what extent the sensitization procedure caused an abnormality of the high-conductance Ca2+-activated K+ channels is unknown. This and other potential mechanisms triggered by the sensitization procedure deserve further investigation. We concluded that during the relaxation induced by 5-HT two major mechanisms seem to be involved: the stimulation of the Na+/K+ATPase pump, and an increased activity of the BKCa channels, both of them probably mediated via 5-HT2A receptors. Acknowledgments This work is part of the PhD degree of Patricia Campos-Bedolla, and we thank Dr. Israel Grijalva for his support in the development of this research. This study was supported by grants from DGAPA-UNAM (IN202107) to Dr. Luis M. Montaño, and from IMSS (2006-1A-I-077) to MSc Patricia Campos-Bedolla. References Adner, M., Rose, A.C., Zhang, Y., Sward, K., Benson, M., Uddman, R., Shankley, N.P., Cardell, L.O., 2002. An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNF-α up-regulates 5HT2A-mediated contraction. British Journal of Pharmacology 137 (7), 971–982. Baron, C.B., Pompeo, J., Blackman, D., Coburn, R.F., 1993. Common phosphatidylinositol 4,5-bisphosphate pools are involved in carbachol and serotonin activation of
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