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New evidence on the mechanisms underlying bradykinin-mediated contraction of the pig iris sphincter in vitro
Peptides 24 (2003) 1045–1051
New evidence on the mechanisms underlying bradykinin-mediated contraction of the pig iris sphincter in vitro Mariem El Sayah, João B. Calixto∗ Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Rua Ferreira Lima 82, Florianópolis, SC 88015-420, Brazil Received 7 March 2003; accepted 3 June 2003
Abstract We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B2 receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8–37), NK2 or NK3 receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK1 antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms. © 2003 Elsevier Inc. All rights reserved. Keywords: Bradykinin; Pig iris sphincter; Calcium channel; Tachykinins; CGRP; Sensory fibers; Capsaicin; Protein kinases A and C
1. Introduction Kinins represent a group of endogenous peptides generated in plasma and peripheral tissues in response to trauma or infection, from their kininogen precursors and by the action of kallikrein enzymes. Kinins exert multiple physiological actions in different tissues, such as smooth muscle contraction or relaxation, control of blood pressure, increase of microvascular leakage, promotion of venular dilatation, mucus secretion, induction of pain and hyperalgesia and they are also involved in inflammatory states [6,7,12,30]. The multiple kinin actions are mediated by the activation of two membrane receptors denoted B1 and B2 . The B2 kinin receptors are constitutively expressed in many cell types and are widely distributed in both the peripheral and central nervous system. They exhibit high affinity for bradykinin (BK) and kallidin, and are responsible for the mediating most of the physiological kinin actions. In contrast, B1 receptors have ∗ Corresponding author. Tel.: +55-48-331-9491/9764; fax: +55-48-222-4164. E-mail addresses: [email protected], [email protected] (J.B. Calixto).
0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0196-9781(03)00182-7
higher affinity for the BK metabolites des-Arg9 -bradykinin and des-Arg10 -kallidin than they do for BK itself [6,7,18,24]. Both B1 and B2 kinin receptors have seven putative transmembrane domains and belong to the family of G-proteincoupled receptors. They have been cloned in several animal species [21,26–28]. The kinin actions are mainly bound to the activation of phospholipase C and to the production of inositol 1,4,5-triphosphate, with consequent increase in intracellular Ca2+ concentrations [25]. It has also been extensively reported that kinin actions are associated with the secondary production of other mediators including prostanoids, tachykinins, cytokines, mast cell-derived products and nitric oxide [6,7]. The infusion of BK or capsaicin into the rabbit’s anterior chambers causes inflammation and pupilar constriction [3,4,8]. It has been reported that BK, like capsaicin, elicits concentration-dependent contraction of the rabbit iris sphincter muscle through the release of substance P from the trigeminal nerve, via activation of B2 receptor [5,11,32,35]. There is also experimental evidence showing that BK contracts rabbit iris sphincter preparations through a tachykinin-dependent mechanism [15,19]. Furthermore, BK applied to the conjuctiva and nasal mucosa of guinea
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pig in vivo causes plasma extravasation and the release of tachykinins from the peripheral endings of the trigeminal nerve, an effect which has been found to be mediated by the activation of B2 receptors [20]. In marked contrast, Geppetti et al. [14] have demonstrated that in the pig iris sphincter (PIS), BK elicites a concentration-dependent contraction through a mechanism which is independent of neuropeptide release from capsaicin-sensitive sensory neurons. In a recent study, we have demonstrated that BK-mediated contraction of the PIS is mediated primarily via activation of the B2 receptor, an action which involves the release of acetylcholine, noradrenaline and both cyclooxygenase-1 and -2 metabolites, besides the release of leukotriene D4 and tromboxane A2 from the arachidonic acid pathway [10]. The present study aimed to characterize further, by the use of selective antagonists and ion channel blockers, some additional mechanisms by which BK mediates contraction in PIS in vitro.
2. Methods 2.1. Tissue preparation Pig eyes were obtained from a local slaughterhouse and immediately transferred in ice to the laboratory. Tissues were used within 6 h after death. Irises were carefully dissected from adherent tissues (two preparations per eye). Each iris was mounted in a 5-ml organ chamber containing Krebs–Henseleit solution of the following composition (in mmol/l): NaCl 118.0; KCl 4.4; CaCl2 2.5; NaHCO3 25.0; MgSO4 1.1; KH2 PO4 1.2, glucose 11.0, maintained at 37 ◦ C, pH 7.4, aerated with gas mixture of 95% O2 and 5% CO2 . Preparations were connected vertically to a force-displacement transducer under a resting tension of 100 mg, which showed the best response and was selected for further experiments [10]. Preparations were allowed to equilibrate for at least 90 min before drug addition and during this period the Krebs–Henseleit solution was changed every 15 min. Isometric contractions were recorded by means of a polygraph (TRI-201 Letica Scientific Instruments, Spain). Usually, four to six preparations (two obtained from each eye) were mounted in parallel. 2.2. Contraction induced by BK After the stabilization period of at least 90 min, and in order to confirm the viability of the tissues, preparations were exposed to a high potassium concentration (80 mM KCl, prepared by equimolar replacement of 74.4 mM of NaCl by KCl) as a standard stimulus. After washout replacement with normal medium and return to the original baseline, complete concentration–response curves (CRCs) were obtained at 60 min intervals for BK (0.1–10 nM). The CRCs were performed by means of the cumulative method [34]. Each con-
centration of the agonist was added to the bath when the effect of the preceding addition had reached its maximum. As reported previously [10], no significant desensitization was observed for any of three consecutive CRCs for BK in the same preparation and no more than three complete curves were obtained for each tissue. The contractile responses for BK are expressed as the percentage of the contraction induced by 80 mM of KCl. 2.3. Additional mechanisms mediated by BK-induced contraction To evaluate the contribution of external Ca2+ in BK-induced contractile responses in the PIS, after 90 min of equilibration period, preparations were transferred for 20 min to Krebs solution without Ca2+ and containing 1 mM of EGTA, during which the bath solution was renewed every 5 min. Responses to BK were subsequently obtained in Ca2+ -free medium and after washout, the preparations were transferred to normal Krebs solution. After a 30 min equilibration period, new CRCs were recorded to assess the recovery of the BK responses. In another set of experiments, after obtaining a CRC for BK, preparations were pre-incubated with one of the following agents— nicardipine (1 M, an antagonist of L-type Ca2+ channel), -conotoxin GVIA, -conotoxin MVIIA (0.1 M, an antagonist of N-type Ca2+ channel) or -agatoxin IVA (0.1 M, antagonist of P-type Ca2+ channel)—20 min before the CRCs for BK were obtained as described above. To assess the role of sensory neuropeptides in BK-mediated contraction, separate preparations were pre-incubated for 30 min with one of the following drugs: 8–37 calcitonin gene-related peptide (CGRP) fragment antagonist (1 M), SR 142801 (NK3 antagonist: 100 nM), SR 48968 (NK2 antagonist: 100 nM) or FK 888 (NK1 antagonist: 100 nM). New CRCs were then obtained in their presence. In protocols examining the possible role of the vanilloid receptor in BK-induced contraction in the PIS, the preparations were exposed to capsaicin (10 M for 30 min) followed by washing and re-equilibration, and a new response for BK was then obtained. In other experiments, preparations were pre-incubated with the selective antagonist of the vanilloid receptor capsazepine (1 M) 20 min prior to obtaining a CRC for BK. To assess the possible contribution of the protein kinases (PK) A and PKC in the BK-mediated contraction of the PIS, after the equilibration period, preparations were pre-incubated with KT 5720 (100 nM) or GF 109203X (1 M) (the PKA and PKC blockers, respectively), and new CRC for BK were obtained in their presence. The concentration of each antagonist and the time of incubation were selected on the basis of data from the literature [1,16,22,23,31,33], or when not available, we performed preliminary experiments to select the best concentration for each of the antagonists used (results not shown).
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2.4. Drugs The following drugs were used: BK, capsaicin, CGRP(8–37), EGTA (all from Sigma Chemical Co., St. Louis, MO, USA), capsazepine (Cayman Chemical Company, Ann Arbor, MI, USA), nicardipine (Research Biochemicals International, Natick, MA, USA), GF 109203X 2-[1-(3dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide, KT 5720 (Tocris Cookson Inc., St. Louis, MO, USA), -agatoxin IVA, -conotoxin GVIA, -conotoxin MVIIA (Alamone Labs, Jerusalem, Israel). The NK2 receptor antagonist SR 48968 (S)-N-methyl-N-[4-(4-acetylamino4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide and the NK3 receptor antagonist SR 142801 (S)-N-(1(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)4-phenylpiperidin-4-yl)-N-methylacetamide were kindly supplied by Sanofi Recherche (Montpellier, France). The NK1 receptor antagonist FK 888 N2 -[(4R)-4-hydroxy1-(1-methyl-1H-indol-3-yl)carbony-l-l-prolyl]-N-methyl-Nphenylmethyl-3-2-(2-naphtyl)-l-alaninamide was supplied by Fujisawa Pharmaceutical Co., Osaka, Japan. The stock solutions of BK were prepared in phosphatebuffered saline (PBS; 1 mM), kept in siliconized plastic tubes and maintained in a freezer at −18 ◦ C until use. Stock solutions of FK 888 were made in absolute ethanol. KT 5720 and GF 109203X were dissolved in dimethyl sulfoxide. All other drugs were dissolved in PBS to the desired concentration just before use. The final bath concentrations of ethanol and dimethyl sulfoxide did not exceed 0.05%. In all experimental groups, at least one parallel control experiment was carried out in the presence of the vehicle used to dilute the drugs. The vehicles used had no pharmacological effects either on the tonus of preparations or on BK-mediated contraction. 2.5. Statistical analysis All values are expressed as means ± S.E.M. Statistical significance was performed by paired or unpaired Student’s t-test. P values less than 0.05 was considered to be statistically significant.
3. Results As described before [10] cumulative addition of BK to PIS produced a concentration-dependent and well-reproducible contractile response with no evidence of tachyphylaxis. To assess the contribution of external Ca2+ to the contractile responses elicited by BK, some experiments were carried out in Ca2+ -free medium containing EGTA (1 mM). Under these conditions, the contractile response caused by BK was totally abolished. When preparations were transferred to normal Krebs solution (containing Ca2+ 2.5 mM) for 30 min, the contraction caused by BK was almost completely re-
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covered (Fig. 1A and results not shown). The results of Fig. 1B–E show that the pre-incubation of the preparations with nicardipine (1 M, an antagonist of L-type Ca2+ channel), -conotoxin GVIA, -conotoxin MVIIA (0.1 M, antagonists of N-type Ca2+ channel) or with -agatoxin IVA (0.1 M, antagonist of P-type Ca2+ channel), produced significant inhibition of the BK-induced contraction in the PIS (33 ± 5%, 29 ± 7%, 63 ± 5% and 36 ± 3%, respectively). The results in Fig. 2B and C show that the SR 48968 (NK2 antagonist: 100 nM) or SR 142801 (NK3 antagonist: 100 nM) receptor antagonists caused significant inhibition of the BK-mediated contraction in the PIS (30 ± 7% and 31 ± 2%, respectively). The FK 888 (NK1 antagonist: 100 nM) produced only a slight, though significant, displacement to the right of the BK-mediated contraction that was associated with reduction of maximal response (23 ± 2%). The 8–37 CGRP fragment antagonist (1 M) also produced a significant inhibition (29 ± 3%) of BK-mediated contraction in PIS (Fig. 2D). In protocols examining the possible role of the vanilloid receptor in BK-induced contraction in the PIS, the preparations were exposed to capsaicin (10 M for 30 min) followed by washing and re-equilibration, and a new response for BK was then obtained. In other experiments, preparations were pre-incubated with the selective antagonist of the vanilloid receptor capsazepine (1 M) 20 min prior obtaining a new CRC for BK. The exposure of the preparations to capsaicin (10 M) for 30 min caused a small though significant inhibition (35 ± 6%) of the contractile response induced by the BK (Fig. 3A). Furthermore, the results in Fig. 3B show that the selective antagonist of the vanilloid receptor capsazepine (1 M) significantly inhibited the contractile response induced by BK (32 ± 5%) in a similar fashion. Additional results show that the pre-incubation of the preparations with the antagonist of the PKA KT 5720 (100 nM) or PKC GF 109203X (1 M) for 30 min caused significant inhibition (42 ± 2% and 37 ± 4%, respectively) of BK-mediated contraction in the PIS (Fig. 4A and B).
4. Discussion In a recent study [10], we demonstrated that the pharmacological activation of kinin B2 receptor subtype is mainly, if not solely, responsible for mediating the contractile response to kinin in the PIS. The BK B1 receptor agonist des-Arg9 -bradykinin only caused a slight contraction, measured 6 h after the tissue was set up, suggesting only a minor role of the constitutive B1 receptor on kinins-mediated contraction in this preparations. Furthermore, we have also shown that BK-mediated contraction in PIS is largely mediated by the neural release of acetylcholine and noradrenaline, since BK-mediated contraction of the PIS is prevented by atropine, guanethidine or tetrodotoxin. Finally, the BK-mediated contraction of the PIS involves the release of prostanoids derived from both cyclooxygenase-1 and -2
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Fig. 1. Cumulative log concentration–response curves for BK in the isolated pig iris obtained in (A) Ca2+ -free solution, (B) nicardipine (1 M), (C) -conotoxin GVIA (0.1 M), (D) -conotoxin MVIIA (0.1 M) or (E) -agatoxin IVA (0.1 M). Results are expressed as percentage of contraction induced by 80 mM of KCl. Each point represents the mean, with vertical lines showing S.E.M. of five experiments. In some points the error deviation is hidden inside the symbol. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
and also the release of leukotriene D4 and tromboxane A2 from arachidonic acid pathway [10]. It has been widely demonstrated that BK B1 and B2 receptors activation is involved in several pathophysiological conditions, and both receptors are involved in the regulation of intracellular Ca2+ [12,18]. The results of the present study extend these previous observations [10] and provide consistent evidence indicating that BK-mediated contraction in PIS is largely, if not exclusively, dependent on the extracellular Ca2+ influx, since its contractile response in this preparation was completely abolished in Ca2+ -free medium containing EGTA. Also relevant are the results demonstrating that several voltage-sensitive Ca2+ channels, namely L-, N- and P-types are probably involved in BK-mediated contraction in PIS. These observations derive from the finding showing that nicardipine (a selective blocker of L-type of Ca2+ channels), -conotoxin GVIA (a selective antagonist of P-type of Ca2+ channels) and -conotoxin MVIIA (a selective antagonist of N-type of Ca2+ ), all at appropriate concentrations, consistently blocked BK-induced contraction in PIS. The possible participation of other Ca2+ channels such as R and T subtypes in BK-mediated contraction in PIS remains to be seen in future studies.
We next investigated the contribution of tachykinin peptides to BK-mediated contraction in the PIS by using selective NK1 , NK2 and NK3 receptor antagonists. Our results show that the release of tachykinin from sensory fibers acting through NK2 and NK3 receptors, and to a lesser extent via NK1 receptors, seems to play a relevant role in BK-mediated contraction in the PIS. Furthermore, BK-mediated contraction in PIS also seems to involve, at least partly, the release of CGRP from sensory neurons, evident by our finding showing that the selective CGRP antagonist, the 8–37 CGRP fragment, significantly antagonized BK-induced contraction in this preparation. To explore further the role played by sensory C fibers in BK-induced contraction in PIS, we desensitized the preparation by using a high concentration of the vanilloid agonist capsaicin or by the pre-incubation of the preparation with the vanilloid (VR1) receptor antagonist capsazepine [22,29,33]. Both procedures were capable of significantly inhibiting BK-mediated contraction in the PIS in vitro. Such findings provide additional evidence supporting the notion that the activation of sensory neurons containing C fibers and the release of neuropeptides such as neurokinins and CGRP participate in BK-mediated contraction in the
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Fig. 2. Cumulative log concentration–response curves obtained for BK in the isolated pig iris obtained in the absence or presence of (A) FK 888 (100 nM), (B) SR 48968 (100 nM), (C) SR 142801 (100 nM) or (D) CGRP (8–37) (1 M). Results are expressed as percentage of contraction induced by 80 mM of KCl. Each point represents the mean, with vertical lines showing S.E.M. of five experiments. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
PIS. Our results are to some extent distinct from those previously reported by Geppetti et al. [14], who demonstrated that BK-mediated contraction in the PIS does not involve the release of neuropeptides from sensory neurons and was
not affected by capsaicin incubation in vitro. However, there are marked differences between the two studies. For instance, our early study [10] demonstrated that the sensitivity (EC50 ) for BK-mediated contraction in the PIS is
Fig. 3. Cumulative log concentration–response curves obtained for BK in the isolated pig iris obtained in preparation dessensitized by capsaicin (10 M) (A) or in presence of capsazepine (1 M) (B). Results are expressed as percentage of contraction induced by 80 mM of KCl; each point represents the mean, with vertical lines showing S.E.M. of five experiments. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
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Fig. 4. Cumulative log concentration–response curves for BK in the isolated pig iris obtained in the absence or presence of (A) KT 5720 (100 nM) or GF 109203X (1 M). Results are expressed as percentage of the contraction induced by 80 mM of KCl. Each point represents the mean, with vertical lines showing S.E.M. of five experiments. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
about 10-fold higher than that reported by Geppetti et al. [14]. Therefore, the action of BK in the PIS is somewhat similar to that reported in the same preparations obtained from rabbit [3–5,17,32,35]. Recent biochemical and pharmacological studies have shown that BK-mediated contraction in most vascular and non-vascular smooth muscles relies largely on extracellular Ca2+ influx and also the activation of phospholipase C, which in turn induces intracellular Ca2+ release and activation of certain PK [2,9,13,36]. Thus, we examined the possible role played by both the PKA and PKC in the BK-mediated contraction of the PIS. The pre-incubation of the preparations with the PKA or PKC inhibitors KT 5720 and GF 109203X, respectively at a concentration known to inhibit both enzymes with some degree of selectivity [23,31], resulted in a significant inhibition of BK-induced contraction, suggesting that BK-mediated contraction in PIS possibly involves the participation of a PKA- and PKC-dependent mechanisms. In the present study, we have also reported additional evidence concerning the mechanisms by which BK-induces contraction in PIS in vitro by demonstrating that its contractile response relies largely on influx of external Ca2+ , an effect that is extremely sensitive to L-, N- and P-types of channel blockers. Furthermore, BK-mediated contraction in PIS is indirectly mediated by activation of C fibers, sensitive to capsaicin and capsazepin, with consequent release of neuropeptides present in sensorial fibers namely tachykinins (mainly via activation of NK2 and NK3 receptors) and CGRP. Finally, BK-mediated contraction in the PIS seems to depend on the activation of both PKA- and PKC-related mechanisms. Taken together this and our previous study [10] suggest that kinins acting through B2 receptors might have an important role in the pig pathophysiology. In this way, it is possible to infer that kinins might have clinical
relevance in the pig ocular system, particularly during tissue injury and inflammatory responses.
Acknowledgments This work was supported by grants from the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), and by the Programa de Apoio aos Núcleos de Excelˆencia (PRONEX) (Brazil). M.E.S. is a Ph.D. student in Pharmacology. We thank the pharmaceutical companies for the kind donation of most of the drugs used in this study. References [1] Belvisi MG, Miura M, Stretton D, Barnes PJ. Capsazepine as a selective antagonist of capsaicin-induced activation of C-fibres in guinea-pig bronchi. Eur J Pharmacol 1992;215:341–4. [2] Besant PG, Tan E, Attwood PV. Mammalian protein histidine kinases. Int J Biochem Cell Biol 2003;35:297–309. [3] Butle JM, Hammond B. Neurogenic responses of the eye to injury. Effect of sensory denervation on the response of the rabbit eye to bradykinin and prostaglandin E1 . Trans Ophthalmol Soc UK 1977;97:668–74. [4] Butler JM, Hammond B. The effects of sensory denervation on the responses of the rabbit eye to prostanglandin E1 , bradykinin and substance P. Br J Pharmacol 1980;69:495–502. [5] Bynke G, Hakanson R, Hörig J, Leander S. Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance P. Eur J Pharmacol 1983;91:469–75. [6] Calixto JB, Cabrini DA, Ferreira J, Campos MM. Kinins in pain and inflammation. Pain 2000;87:1–5. [7] Calixto JB, Cabrini DA, Ferreira J, Campos MM. Inflammatory pain: kinins and antagonists. Curr Opin Anaesth 2001;34:314– 22. [8] Cole DF, Unger WG. Action of bradykinin on intraocular pressure and pupillary diameter. Ophthalmic Res 1974;6:308–14.
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New evidence on the mechanisms underlying bradykinin-mediated contraction of the pig iris sphincter in vitro Mariem El Sayah, João B. Calixto∗ Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Rua Ferreira Lima 82, Florianópolis, SC 88015-420, Brazil Received 7 March 2003; accepted 3 June 2003
Abstract We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B2 receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8–37), NK2 or NK3 receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK1 antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms. © 2003 Elsevier Inc. All rights reserved. Keywords: Bradykinin; Pig iris sphincter; Calcium channel; Tachykinins; CGRP; Sensory fibers; Capsaicin; Protein kinases A and C
1. Introduction Kinins represent a group of endogenous peptides generated in plasma and peripheral tissues in response to trauma or infection, from their kininogen precursors and by the action of kallikrein enzymes. Kinins exert multiple physiological actions in different tissues, such as smooth muscle contraction or relaxation, control of blood pressure, increase of microvascular leakage, promotion of venular dilatation, mucus secretion, induction of pain and hyperalgesia and they are also involved in inflammatory states [6,7,12,30]. The multiple kinin actions are mediated by the activation of two membrane receptors denoted B1 and B2 . The B2 kinin receptors are constitutively expressed in many cell types and are widely distributed in both the peripheral and central nervous system. They exhibit high affinity for bradykinin (BK) and kallidin, and are responsible for the mediating most of the physiological kinin actions. In contrast, B1 receptors have ∗ Corresponding author. Tel.: +55-48-331-9491/9764; fax: +55-48-222-4164. E-mail addresses: [email protected], [email protected] (J.B. Calixto).
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higher affinity for the BK metabolites des-Arg9 -bradykinin and des-Arg10 -kallidin than they do for BK itself [6,7,18,24]. Both B1 and B2 kinin receptors have seven putative transmembrane domains and belong to the family of G-proteincoupled receptors. They have been cloned in several animal species [21,26–28]. The kinin actions are mainly bound to the activation of phospholipase C and to the production of inositol 1,4,5-triphosphate, with consequent increase in intracellular Ca2+ concentrations [25]. It has also been extensively reported that kinin actions are associated with the secondary production of other mediators including prostanoids, tachykinins, cytokines, mast cell-derived products and nitric oxide [6,7]. The infusion of BK or capsaicin into the rabbit’s anterior chambers causes inflammation and pupilar constriction [3,4,8]. It has been reported that BK, like capsaicin, elicits concentration-dependent contraction of the rabbit iris sphincter muscle through the release of substance P from the trigeminal nerve, via activation of B2 receptor [5,11,32,35]. There is also experimental evidence showing that BK contracts rabbit iris sphincter preparations through a tachykinin-dependent mechanism [15,19]. Furthermore, BK applied to the conjuctiva and nasal mucosa of guinea
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pig in vivo causes plasma extravasation and the release of tachykinins from the peripheral endings of the trigeminal nerve, an effect which has been found to be mediated by the activation of B2 receptors [20]. In marked contrast, Geppetti et al. [14] have demonstrated that in the pig iris sphincter (PIS), BK elicites a concentration-dependent contraction through a mechanism which is independent of neuropeptide release from capsaicin-sensitive sensory neurons. In a recent study, we have demonstrated that BK-mediated contraction of the PIS is mediated primarily via activation of the B2 receptor, an action which involves the release of acetylcholine, noradrenaline and both cyclooxygenase-1 and -2 metabolites, besides the release of leukotriene D4 and tromboxane A2 from the arachidonic acid pathway [10]. The present study aimed to characterize further, by the use of selective antagonists and ion channel blockers, some additional mechanisms by which BK mediates contraction in PIS in vitro.
2. Methods 2.1. Tissue preparation Pig eyes were obtained from a local slaughterhouse and immediately transferred in ice to the laboratory. Tissues were used within 6 h after death. Irises were carefully dissected from adherent tissues (two preparations per eye). Each iris was mounted in a 5-ml organ chamber containing Krebs–Henseleit solution of the following composition (in mmol/l): NaCl 118.0; KCl 4.4; CaCl2 2.5; NaHCO3 25.0; MgSO4 1.1; KH2 PO4 1.2, glucose 11.0, maintained at 37 ◦ C, pH 7.4, aerated with gas mixture of 95% O2 and 5% CO2 . Preparations were connected vertically to a force-displacement transducer under a resting tension of 100 mg, which showed the best response and was selected for further experiments [10]. Preparations were allowed to equilibrate for at least 90 min before drug addition and during this period the Krebs–Henseleit solution was changed every 15 min. Isometric contractions were recorded by means of a polygraph (TRI-201 Letica Scientific Instruments, Spain). Usually, four to six preparations (two obtained from each eye) were mounted in parallel. 2.2. Contraction induced by BK After the stabilization period of at least 90 min, and in order to confirm the viability of the tissues, preparations were exposed to a high potassium concentration (80 mM KCl, prepared by equimolar replacement of 74.4 mM of NaCl by KCl) as a standard stimulus. After washout replacement with normal medium and return to the original baseline, complete concentration–response curves (CRCs) were obtained at 60 min intervals for BK (0.1–10 nM). The CRCs were performed by means of the cumulative method [34]. Each con-
centration of the agonist was added to the bath when the effect of the preceding addition had reached its maximum. As reported previously [10], no significant desensitization was observed for any of three consecutive CRCs for BK in the same preparation and no more than three complete curves were obtained for each tissue. The contractile responses for BK are expressed as the percentage of the contraction induced by 80 mM of KCl. 2.3. Additional mechanisms mediated by BK-induced contraction To evaluate the contribution of external Ca2+ in BK-induced contractile responses in the PIS, after 90 min of equilibration period, preparations were transferred for 20 min to Krebs solution without Ca2+ and containing 1 mM of EGTA, during which the bath solution was renewed every 5 min. Responses to BK were subsequently obtained in Ca2+ -free medium and after washout, the preparations were transferred to normal Krebs solution. After a 30 min equilibration period, new CRCs were recorded to assess the recovery of the BK responses. In another set of experiments, after obtaining a CRC for BK, preparations were pre-incubated with one of the following agents— nicardipine (1 M, an antagonist of L-type Ca2+ channel), -conotoxin GVIA, -conotoxin MVIIA (0.1 M, an antagonist of N-type Ca2+ channel) or -agatoxin IVA (0.1 M, antagonist of P-type Ca2+ channel)—20 min before the CRCs for BK were obtained as described above. To assess the role of sensory neuropeptides in BK-mediated contraction, separate preparations were pre-incubated for 30 min with one of the following drugs: 8–37 calcitonin gene-related peptide (CGRP) fragment antagonist (1 M), SR 142801 (NK3 antagonist: 100 nM), SR 48968 (NK2 antagonist: 100 nM) or FK 888 (NK1 antagonist: 100 nM). New CRCs were then obtained in their presence. In protocols examining the possible role of the vanilloid receptor in BK-induced contraction in the PIS, the preparations were exposed to capsaicin (10 M for 30 min) followed by washing and re-equilibration, and a new response for BK was then obtained. In other experiments, preparations were pre-incubated with the selective antagonist of the vanilloid receptor capsazepine (1 M) 20 min prior to obtaining a CRC for BK. To assess the possible contribution of the protein kinases (PK) A and PKC in the BK-mediated contraction of the PIS, after the equilibration period, preparations were pre-incubated with KT 5720 (100 nM) or GF 109203X (1 M) (the PKA and PKC blockers, respectively), and new CRC for BK were obtained in their presence. The concentration of each antagonist and the time of incubation were selected on the basis of data from the literature [1,16,22,23,31,33], or when not available, we performed preliminary experiments to select the best concentration for each of the antagonists used (results not shown).
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2.4. Drugs The following drugs were used: BK, capsaicin, CGRP(8–37), EGTA (all from Sigma Chemical Co., St. Louis, MO, USA), capsazepine (Cayman Chemical Company, Ann Arbor, MI, USA), nicardipine (Research Biochemicals International, Natick, MA, USA), GF 109203X 2-[1-(3dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide, KT 5720 (Tocris Cookson Inc., St. Louis, MO, USA), -agatoxin IVA, -conotoxin GVIA, -conotoxin MVIIA (Alamone Labs, Jerusalem, Israel). The NK2 receptor antagonist SR 48968 (S)-N-methyl-N-[4-(4-acetylamino4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide and the NK3 receptor antagonist SR 142801 (S)-N-(1(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)4-phenylpiperidin-4-yl)-N-methylacetamide were kindly supplied by Sanofi Recherche (Montpellier, France). The NK1 receptor antagonist FK 888 N2 -[(4R)-4-hydroxy1-(1-methyl-1H-indol-3-yl)carbony-l-l-prolyl]-N-methyl-Nphenylmethyl-3-2-(2-naphtyl)-l-alaninamide was supplied by Fujisawa Pharmaceutical Co., Osaka, Japan. The stock solutions of BK were prepared in phosphatebuffered saline (PBS; 1 mM), kept in siliconized plastic tubes and maintained in a freezer at −18 ◦ C until use. Stock solutions of FK 888 were made in absolute ethanol. KT 5720 and GF 109203X were dissolved in dimethyl sulfoxide. All other drugs were dissolved in PBS to the desired concentration just before use. The final bath concentrations of ethanol and dimethyl sulfoxide did not exceed 0.05%. In all experimental groups, at least one parallel control experiment was carried out in the presence of the vehicle used to dilute the drugs. The vehicles used had no pharmacological effects either on the tonus of preparations or on BK-mediated contraction. 2.5. Statistical analysis All values are expressed as means ± S.E.M. Statistical significance was performed by paired or unpaired Student’s t-test. P values less than 0.05 was considered to be statistically significant.
3. Results As described before [10] cumulative addition of BK to PIS produced a concentration-dependent and well-reproducible contractile response with no evidence of tachyphylaxis. To assess the contribution of external Ca2+ to the contractile responses elicited by BK, some experiments were carried out in Ca2+ -free medium containing EGTA (1 mM). Under these conditions, the contractile response caused by BK was totally abolished. When preparations were transferred to normal Krebs solution (containing Ca2+ 2.5 mM) for 30 min, the contraction caused by BK was almost completely re-
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covered (Fig. 1A and results not shown). The results of Fig. 1B–E show that the pre-incubation of the preparations with nicardipine (1 M, an antagonist of L-type Ca2+ channel), -conotoxin GVIA, -conotoxin MVIIA (0.1 M, antagonists of N-type Ca2+ channel) or with -agatoxin IVA (0.1 M, antagonist of P-type Ca2+ channel), produced significant inhibition of the BK-induced contraction in the PIS (33 ± 5%, 29 ± 7%, 63 ± 5% and 36 ± 3%, respectively). The results in Fig. 2B and C show that the SR 48968 (NK2 antagonist: 100 nM) or SR 142801 (NK3 antagonist: 100 nM) receptor antagonists caused significant inhibition of the BK-mediated contraction in the PIS (30 ± 7% and 31 ± 2%, respectively). The FK 888 (NK1 antagonist: 100 nM) produced only a slight, though significant, displacement to the right of the BK-mediated contraction that was associated with reduction of maximal response (23 ± 2%). The 8–37 CGRP fragment antagonist (1 M) also produced a significant inhibition (29 ± 3%) of BK-mediated contraction in PIS (Fig. 2D). In protocols examining the possible role of the vanilloid receptor in BK-induced contraction in the PIS, the preparations were exposed to capsaicin (10 M for 30 min) followed by washing and re-equilibration, and a new response for BK was then obtained. In other experiments, preparations were pre-incubated with the selective antagonist of the vanilloid receptor capsazepine (1 M) 20 min prior obtaining a new CRC for BK. The exposure of the preparations to capsaicin (10 M) for 30 min caused a small though significant inhibition (35 ± 6%) of the contractile response induced by the BK (Fig. 3A). Furthermore, the results in Fig. 3B show that the selective antagonist of the vanilloid receptor capsazepine (1 M) significantly inhibited the contractile response induced by BK (32 ± 5%) in a similar fashion. Additional results show that the pre-incubation of the preparations with the antagonist of the PKA KT 5720 (100 nM) or PKC GF 109203X (1 M) for 30 min caused significant inhibition (42 ± 2% and 37 ± 4%, respectively) of BK-mediated contraction in the PIS (Fig. 4A and B).
4. Discussion In a recent study [10], we demonstrated that the pharmacological activation of kinin B2 receptor subtype is mainly, if not solely, responsible for mediating the contractile response to kinin in the PIS. The BK B1 receptor agonist des-Arg9 -bradykinin only caused a slight contraction, measured 6 h after the tissue was set up, suggesting only a minor role of the constitutive B1 receptor on kinins-mediated contraction in this preparations. Furthermore, we have also shown that BK-mediated contraction in PIS is largely mediated by the neural release of acetylcholine and noradrenaline, since BK-mediated contraction of the PIS is prevented by atropine, guanethidine or tetrodotoxin. Finally, the BK-mediated contraction of the PIS involves the release of prostanoids derived from both cyclooxygenase-1 and -2
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Fig. 1. Cumulative log concentration–response curves for BK in the isolated pig iris obtained in (A) Ca2+ -free solution, (B) nicardipine (1 M), (C) -conotoxin GVIA (0.1 M), (D) -conotoxin MVIIA (0.1 M) or (E) -agatoxin IVA (0.1 M). Results are expressed as percentage of contraction induced by 80 mM of KCl. Each point represents the mean, with vertical lines showing S.E.M. of five experiments. In some points the error deviation is hidden inside the symbol. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
and also the release of leukotriene D4 and tromboxane A2 from arachidonic acid pathway [10]. It has been widely demonstrated that BK B1 and B2 receptors activation is involved in several pathophysiological conditions, and both receptors are involved in the regulation of intracellular Ca2+ [12,18]. The results of the present study extend these previous observations [10] and provide consistent evidence indicating that BK-mediated contraction in PIS is largely, if not exclusively, dependent on the extracellular Ca2+ influx, since its contractile response in this preparation was completely abolished in Ca2+ -free medium containing EGTA. Also relevant are the results demonstrating that several voltage-sensitive Ca2+ channels, namely L-, N- and P-types are probably involved in BK-mediated contraction in PIS. These observations derive from the finding showing that nicardipine (a selective blocker of L-type of Ca2+ channels), -conotoxin GVIA (a selective antagonist of P-type of Ca2+ channels) and -conotoxin MVIIA (a selective antagonist of N-type of Ca2+ ), all at appropriate concentrations, consistently blocked BK-induced contraction in PIS. The possible participation of other Ca2+ channels such as R and T subtypes in BK-mediated contraction in PIS remains to be seen in future studies.
We next investigated the contribution of tachykinin peptides to BK-mediated contraction in the PIS by using selective NK1 , NK2 and NK3 receptor antagonists. Our results show that the release of tachykinin from sensory fibers acting through NK2 and NK3 receptors, and to a lesser extent via NK1 receptors, seems to play a relevant role in BK-mediated contraction in the PIS. Furthermore, BK-mediated contraction in PIS also seems to involve, at least partly, the release of CGRP from sensory neurons, evident by our finding showing that the selective CGRP antagonist, the 8–37 CGRP fragment, significantly antagonized BK-induced contraction in this preparation. To explore further the role played by sensory C fibers in BK-induced contraction in PIS, we desensitized the preparation by using a high concentration of the vanilloid agonist capsaicin or by the pre-incubation of the preparation with the vanilloid (VR1) receptor antagonist capsazepine [22,29,33]. Both procedures were capable of significantly inhibiting BK-mediated contraction in the PIS in vitro. Such findings provide additional evidence supporting the notion that the activation of sensory neurons containing C fibers and the release of neuropeptides such as neurokinins and CGRP participate in BK-mediated contraction in the
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Fig. 2. Cumulative log concentration–response curves obtained for BK in the isolated pig iris obtained in the absence or presence of (A) FK 888 (100 nM), (B) SR 48968 (100 nM), (C) SR 142801 (100 nM) or (D) CGRP (8–37) (1 M). Results are expressed as percentage of contraction induced by 80 mM of KCl. Each point represents the mean, with vertical lines showing S.E.M. of five experiments. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
PIS. Our results are to some extent distinct from those previously reported by Geppetti et al. [14], who demonstrated that BK-mediated contraction in the PIS does not involve the release of neuropeptides from sensory neurons and was
not affected by capsaicin incubation in vitro. However, there are marked differences between the two studies. For instance, our early study [10] demonstrated that the sensitivity (EC50 ) for BK-mediated contraction in the PIS is
Fig. 3. Cumulative log concentration–response curves obtained for BK in the isolated pig iris obtained in preparation dessensitized by capsaicin (10 M) (A) or in presence of capsazepine (1 M) (B). Results are expressed as percentage of contraction induced by 80 mM of KCl; each point represents the mean, with vertical lines showing S.E.M. of five experiments. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
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Fig. 4. Cumulative log concentration–response curves for BK in the isolated pig iris obtained in the absence or presence of (A) KT 5720 (100 nM) or GF 109203X (1 M). Results are expressed as percentage of the contraction induced by 80 mM of KCl. Each point represents the mean, with vertical lines showing S.E.M. of five experiments. Significant differences from respective control values where ∗ P < 0.05 (Student’s unpaired t-test).
about 10-fold higher than that reported by Geppetti et al. [14]. Therefore, the action of BK in the PIS is somewhat similar to that reported in the same preparations obtained from rabbit [3–5,17,32,35]. Recent biochemical and pharmacological studies have shown that BK-mediated contraction in most vascular and non-vascular smooth muscles relies largely on extracellular Ca2+ influx and also the activation of phospholipase C, which in turn induces intracellular Ca2+ release and activation of certain PK [2,9,13,36]. Thus, we examined the possible role played by both the PKA and PKC in the BK-mediated contraction of the PIS. The pre-incubation of the preparations with the PKA or PKC inhibitors KT 5720 and GF 109203X, respectively at a concentration known to inhibit both enzymes with some degree of selectivity [23,31], resulted in a significant inhibition of BK-induced contraction, suggesting that BK-mediated contraction in PIS possibly involves the participation of a PKA- and PKC-dependent mechanisms. In the present study, we have also reported additional evidence concerning the mechanisms by which BK-induces contraction in PIS in vitro by demonstrating that its contractile response relies largely on influx of external Ca2+ , an effect that is extremely sensitive to L-, N- and P-types of channel blockers. Furthermore, BK-mediated contraction in PIS is indirectly mediated by activation of C fibers, sensitive to capsaicin and capsazepin, with consequent release of neuropeptides present in sensorial fibers namely tachykinins (mainly via activation of NK2 and NK3 receptors) and CGRP. Finally, BK-mediated contraction in the PIS seems to depend on the activation of both PKA- and PKC-related mechanisms. Taken together this and our previous study [10] suggest that kinins acting through B2 receptors might have an important role in the pig pathophysiology. In this way, it is possible to infer that kinins might have clinical
relevance in the pig ocular system, particularly during tissue injury and inflammatory responses.
Acknowledgments This work was supported by grants from the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), and by the Programa de Apoio aos Núcleos de Excelˆencia (PRONEX) (Brazil). M.E.S. is a Ph.D. student in Pharmacology. We thank the pharmaceutical companies for the kind donation of most of the drugs used in this study. References [1] Belvisi MG, Miura M, Stretton D, Barnes PJ. Capsazepine as a selective antagonist of capsaicin-induced activation of C-fibres in guinea-pig bronchi. Eur J Pharmacol 1992;215:341–4. [2] Besant PG, Tan E, Attwood PV. Mammalian protein histidine kinases. Int J Biochem Cell Biol 2003;35:297–309. [3] Butle JM, Hammond B. Neurogenic responses of the eye to injury. Effect of sensory denervation on the response of the rabbit eye to bradykinin and prostaglandin E1 . Trans Ophthalmol Soc UK 1977;97:668–74. [4] Butler JM, Hammond B. The effects of sensory denervation on the responses of the rabbit eye to prostanglandin E1 , bradykinin and substance P. Br J Pharmacol 1980;69:495–502. [5] Bynke G, Hakanson R, Hörig J, Leander S. Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance P. Eur J Pharmacol 1983;91:469–75. [6] Calixto JB, Cabrini DA, Ferreira J, Campos MM. Kinins in pain and inflammation. Pain 2000;87:1–5. [7] Calixto JB, Cabrini DA, Ferreira J, Campos MM. Inflammatory pain: kinins and antagonists. Curr Opin Anaesth 2001;34:314– 22. [8] Cole DF, Unger WG. Action of bradykinin on intraocular pressure and pupillary diameter. Ophthalmic Res 1974;6:308–14.
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