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(Investigative Ophthalmology and Visual Science. 2000;41:1861-1870.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Mechanisms Mediating Substance P–Induced Contraction in the Rat Iris In Vitro

Astor Grumann-Júnior, Marcos Antônio Dias, Ricardo Vieira Alves, Joel E. Boteon and João Batista Calixto

From the Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Florianópolis, Brazil.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To determine some of the mechanisms by which substance P (SP) induces contraction in the isolated rat iris.

METHODS. Rings of rat iris were mounted in a 5-ml organ chamber containing Krebs solution at 37°C under basal tension of 75 mg, and isometric tension was recorded.

RESULTS. Substance P produced graded contraction in the rat iris, being approximately 40-fold more potent than carbachol. Peptidase inhibitors (captopril, phosphoramidon, thiorphan) did not affect the SP response. The SP contraction was dependent on external Ca2+ by a mechanism resistant to both nifedipine and {omega}-conotoxin GVIA. Atropine and tetrodotoxin significantly shifted the SP response to the right (three- and fivefold, respectively). Neither phorbol nor genistein altered the SP-induced contraction, whereas staurosporine caused a weak inhibition. Indomethacin, pyrilamine, guanethidine, 8–37 calcitonin gene–related peptide (CGRP) fragment, and NG-nitro-L-arginine methyl ester had no effect on SP response. All the natural tachykinin agonists caused concentration-dependent contraction in rat iris with similar maximal responses. The NK3 selective agonist senktide caused graded contraction, being approximately 150-fold more active than the NK2 selective agonist [ß-ala] NKA. The NK1 selective agonist SP methyl ester induced a small contraction. The NK3 and NK2 antagonists SR 142801 and SR 48968 shifted the SP response to the right. Schilds plots gave pA2 (negative logarithm of the molar concentration of antagonist causing a twofold rightward displacement of the concentration response curves) values of 9.37 and 7.97 and slopes of 0.70 and 1.02, respectively.

CONCLUSIONS. Substance P produces a potent contraction in the isolated rat iris that seems to depend on the neural release of acetylcholine by tetrodotoxin-sensitive mechanisms. Its response relies largely on external Ca2+, through mechanisms independent of activation of L- or N-type Ca2+ channels, and is probably mediated via activation of NK3 and NK2 receptors.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The nonadrenergic, noncholinergic response of the iris has been investigated in recent years, and many kinds of neurotransmitter substances have been described in this process. Since the early 1980s, when specific and high affinity binding sites for substance P (SP) and other tachykinins such as neurokinin A (NKA) and neurokinin B (NKB) were found in the rabbit iris,1 2 3 4 the role played by tachykinins in the iris responsiveness has been studied in different animal species. In rabbits, the most studied species, the actions of some neuropeptides, including SP, have been well established as very powerful stimulating agents.5

The tachykinins are a family of neuropeptides widely distributed in the mammalian central and peripheral nervous systems that produce a wide range of biological effects through the stimulation of at least three distinct receptor types: NK1, NK2, and NK3.6 Molecular cloning studies have recently revealed that all of the three subtypes of tachykinin receptors are members of the seven transmembrane G protein–coupled receptor family.7 8 The endogenous tachykinins substance P (SP), NKA, and NKB bind preferentially, but not exclusively, at NK1, NK2, and NK3 receptors, respectively.

Ocular injury to the rabbit eye results in symptoms of neurogenic inflammation characterized by vasodilatation, plasma extravasation, breakdown of the blood-aqueous barrier, and miosis. There is now evidence suggesting that tachykinins are involved in these responses as a consequence of antidromic reflexes in sensory fibers originating in the trigeminal ganglion.4 9 10 11 In addition, the SP antagonists were found to be capable of abolishing the response to ocular trauma.12 Additional evidence for the involvement of tachykinins in the ocular system is suggested by the presence of NK1 and NK3 receptors in rabbit iris sphincter.13 Furthermore, it has been reported that tachykinin response in the rabbit iris is mediated mainly through the activation of NK3 receptor.14

Few pharmacological studies have been carried out on other animal species. However, differences in tachykinin responsiveness have been reported among them.15 In the rat iris, it has been observed that exogenous administration of SP produces a concentration-dependent contractile response comparable to that produced by acetylcholine.16 However, to the best of our knowledge, no pharmacological study has been performed to characterize the mechanisms by which tachykinin induces contractile responses in the rat iris.

The present study was therefore designed to characterize pharmacologically some of the mechanisms by which SP induces contraction in the rat iris "in vitro." For this, SP and other natural and synthetic tachykinergic agonists were used, aiming to investigate the possible interaction with other mediators (acetylcholine, noradrenaline, prostanoids) and also to analyze the participation of external Ca2+, nitric oxide (NO), and second messenger mechanisms in the SP-mediated contractile response. Finally, the tachykinin receptor type(s) involved in this process were also investigated by the use of highly selective natural and synthetic agonists and antagonists.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Tissue Preparation
Male Wistar rats (300–350 g), cared for and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, maintained under a 12-hour light/dark cycle at 22°C ± 2°C and fed with a standard commercial diet, were killed with a blow on the neck followed by cervical dislocation.

The iris was rapidly removed under optic microscopy (DF Vasconcelos, São Paulo, Brazil), placed in a petri dish containing warmed Krebs–Henseleit solution (see composition below), oxygenated, and maintained at 37°C. The irises were carefully dissected from adherent tissues (two preparations per animal) and were tied with 8-0 silk. Each iris was mounted in a 5-ml organ chamber containing Krebs–Henseleit solution, maintained at 37°C at pH 7.4, which was continuously aerated with a gas mixture of 95% O2 and 5% CO2. The Krebs solution had the following composition (in millimoles): NaCl 118.0, KCl 4.4, MgSO4 1.1, CaCl2 2.5, NaHCO3 25.0, KH2PO4 1.2, and glucose 11.0. The preparation was connected vertically to a force-displacement transducer under a resting tension of 75 mg. The optimal tension was adjusted according to the basis established in preliminary experiments. Preparations were allowed to equilibrate for 90 minutes before the addition of drug, which in preliminary experiments showed the optimal time, during which the bath solution was renewed every 15 minutes. Isometric contractions were recorded by means of a polygraph (TRI–201 Letica Scientific Instruments, Barcelona, Spain). The contractile responses to SP and other agonists are expressed in milligrams of tension developed per preparation. Usually, 4 to 6 preparations were used in parallel. In all experiments, at least one preparation received only the agonist tested and served as control.

Concentration-Response Curves for SP and Other Tachykinins
After the stabilization period of 90 minutes, complete concentration-response curves (CRCs) were obtained for SP (0.1–1000 nM) and related tachykinins (NKA, 0.1–1000 nM; [ß-ala] NKA, 4–10, 0.1–1000 nM; SP methyl-ester, 0.1–1000 nM; NKB, 0.001–1000 nM; and senktide, 0.001–1000 nM) at 60-minute intervals. The CRCs for all studied agonists were performed by means of the cumulative method. Each concentration of the agonist was added to the bath when the effect of the preceding addition had reached its maximum. No significant desensitization was observed for at least three consecutive CRCs for SP in the same preparation. Accordingly, no more than 3 complete curves were carried out in each tissue.

The possible role played by proteases was assessed by preincubation of the preparations with thiorphan (an enkephalinase inhibitor; 10 µM), phospharamidon (an enkephalinase inhibitor; 10 µM), and captopril (angiotensin-converting enzyme inhibitor; 10 µM) 30 minutes beforehand, and, subsequently, SP curves were obtained in the absence or presence of these protease inhibitors, alone or in association.

To assess the possible contribution of external Ca2+ in SP-induced contractile responses in the rat iris, after 90 minutes of equilibration, preparations were transferred to Krebs solution without Ca2+, containing 1 mM EGTA, for 20 minutes, during which the bath solution was renewed every 5 minutes, and responses to SP were subsequently obtained in Ca2+-free medium. After washout, the preparations were transferred to normal Krebs solution, and after 30 minutes of equilibration new contractile responses were recorded to assess the recovery of the SP responses. In another set of experiments, preparations were preincubated for 90 minutes, and SP responses were obtained either in the absence or the presence of nifedipine (1 µM), {omega}-conotoxin GVIA (0,1 µM), or nickel (1 mM; all incubated 20 minutes beforehand).

To investigate the participation of calcitonin gene–related peptide (CGRP) in SP-mediated responses, after 90 minutes of equilibration, cumulative CRCs were obtained for CGRP in the isolated rat iris. After washout and 60 minutes of equilibration, another complete CRC was obtained for SP in the presence of CGRP (30 µM). To further examine the role of CGRP in SP-mediated contraction in the rat iris, preparations were preincubated with CGRP antagonists (8–37; 1 µM), and a new complete CRC was obtained from SP in its presence.

A separate set of experiments was designed to evaluate the participation of protein kinase C and tyrosine kinase in SP-mediated contraction in rat iris. After the equilibration period, preparations were preincubated with staurosporine (1 µM, a protein kinase C antagonist), phorbol 12-myristate 13-acetate (10 µM, an activator of protein kinase C), or genistein (1 µM, an inhibitor of tyrosine kinase), and, 30 minutes afterward, complete CRCs were obtained for SP in their presence.

In a separate set of experiments we assessed the role played by NO in the SP-induced contraction in the rat iris. After the equilibration period, complete CRCs for SP were performed in which NG-nitro-L-arginine methyl ester (L-NAME; 1 µM) was either absent or was added to the bath 30 minutes prior.

The participation of other neurotransmitters in SP-mediated rat iris contraction was also investigated. Preparations were treated with one of the following agents: atropine (an anticholinergic agent; 1 µM), pyrilamine (an antihistaminic agent; 1 µM), tetrodotoxin (TTX; a sodium channel blocker; 1 µM), indomethacin (a cyclo-oxygenase inhibitor; 1 µM), or guanethidine (a norepinephrine depletor; 1 µM). All drugs were preincubated with the tissues 20 minutes beforehand. Only one kind of antagonist was used in each experiment.

In a separate series of experiments, after 90 minutes of equilibration, complete CRCs were obtained for SP (0.1–1000 nM), in the absence or presence of SR 142801 (an NK3 antagonist; 1–10 nM) or SR 48968 (an NK2 antagonist; 10–100 nM). The antagonists were added to the preparation at least 30 minutes before challenge with the agonist. Responses in the absence or presence of the antagonists were expressed in milligrams of tension. Regressions of log (DR-1) against log [B], where DR is the ratio of EC50 value in the presence of a concentration of antagonist divided by the EC50 value in the absence of antagonist and [B] is the molar concentration of antagonist, were plotted according to the method described by Arunlakshana and Schilds (in 1959).17 Least-square regression analysis was used to obtain the slope of the line of best fit for the combined points from a number of experiments. The pA2 values were determined as the negative logarithm of the molar concentration of antagonist causing a twofold rightward displacement of the CRC to a given agonist.

Statistical Analysis
All values are expressed as the mean ± SEM, except the EC50 values (i.e., the molar concentration of the drugs required to produce 50% of the maximal response) and the slopes of the Schilds regression lines, which are reported as the geometric means accompanied by 95% confidence limits. The EC50 values were calculated by means of linear regression analysis from complete CRCs in individual experiments. Tests for statistical significance were performed using Student’s t-test, either paired or unpaired. P < 0.05 or less was considered as indicative of significance. The corresponding test used is indicated below each figure.

Drugs
The following drugs were used: SP, SP methyl ester, ß-ala NKA, senkitide, NKA, NKB (Peninsula, Belmont, CA), CGRP 8-37, L-NAME, staurosporine, tetrodotoxin, indomethacin, guanethidine, phospharamidon, thiorphan, phorbol 12-myristate 13-acetate, pyrilamine, genistein, carbachol, nickel hydrate, {omega}-conotoxin GVIA, EGTA (all from Sigma Chemical, St. Louis, MO), captopril, NKA, verapamil, nifedipine (Research Biochemicals International, Natick, MA), and atropine sulfate (E. Merck, Darmstadt, Germany). The NK3 and NK2 receptor antagonists SR 142801, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide, and SR 48968, (S)-N-methyl-N[4-(4acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide, were kindly provided by Sanofi Recherché (Montpellier, France).

The stock solutions for all peptides used were prepared in phosphate-buffered saline (PBS; 1–10 mM), kept in siliconized plastic tubes, and maintained in a freezer at -18°C until use. Stock solutions of indomethacin, staurosporine, and phorbol 12-myristate 13-acetate were made in absolute ethanol, but the final concentration of ethanol did not exceed 0.05%, to avoid effects on either SP-induced contraction or the tone of the preparations. All other drugs were dissolved in PBS to the desired concentration just before use.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Characterization of the Contractile Response Induced by SP in Rat Iris
SP produced a concentration-dependent contraction in the isolated rat iris with mean EC50 (and 95% confidence limits) of 23.09 (12.42–42.94) nM and maximal response (Emax) of 43.33 (±5.53) mg of tension (Fig. 1) . At the EC50 level, SP was approximately 40 times more potent than carbachol, with EC50 (and 95% confidence limits) of 941.21 (562.91–1573.22) nM, although the Emax 47.02 (±6.75) mg was similar to that of SP (Fig. 1) . As mentioned in the Methods section, no tachyphylaxis for SP contractile responses was observed when experiments were carried out at 60-minute intervals between curves (results not shown).



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  		Figure 1. Cumulative log concentration-effect for SP and carbachol in the isolated rat iris. Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing SEM of 5 experiments.

 
The preincubation of the preparations with the protease inhibitors (captopril 1 µM, phosphoramidom 1 µM, and thiorphan 1 µM), either alone or in association, did not change the tonus of the preparations and also failed to significantly affect the contractile response induced by SP (results not shown).

Influence of Atropine, TTX, and Other Antagonists on SP-Mediated Contraction
Preincubation of the rat iris with atropine (1 µM) 20 minutes beforehand produced marked (approximately 3.5-fold) rightward displacement of the SP-induced contraction without affecting its Emax (Fig. 2A ). The addition of TTX (1 µM) to the preparations 20 minutes before also produced a significant (about fivefold) displacement to the right of the SP-induced contraction in the rat iris without changing the Emax (Fig. 2B) . On the other hand, preincubation with indomethacin (1 µM), guanethidine (1 µM), or pyrilamine (1 µM) for 20 minutes had no significant effect on SP-induced contraction of the rat iris (Table 1) .



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  		Figure 2. Cumulative log CRCs for SP in the isolated rat iris obtained in the absence or presence of (A) atropine (1 µM) and (B) TTX (1 µM). Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing SEM of 5 experiments. Significant differences from respective control values.

 

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  		Table 1. Effect of Several Classes of Drugs (1 µM) on the Concentration-Dependent Response Curve Induced by SP (0.1–1000 nM) in the Isolated Rat Iris

 
Influence of Extracellular Calcium on SP-Mediated Contraction
To assess the contribution of external Ca2+ in the contractile responses elicited by SP, some experiments were carried out in Ca2+-free medium containing EGTA (1 mM). Under these conditions, there was a marked reduction (79.49% ± 4.90%) of the contraction induced by SP (100 nM) compared with experiments performed in normal Krebs solution (P < 0.05; Fig. 3 ). When preparations were transferred to normal Krebs solution (Ca2+ 2.5 mM) for 30 minutes, the contraction caused by SP was almost completely recovered (87.90% ± 13.12%; Fig. 3 ).



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  		Figure 3. Effect of external Ca2+ on the SP (10 µM)–mediated contraction of isolated rat iris. Control responses and responses obtained in preparations maintained in calcium-free medium containing EGTA (1 mM). Thirty minutes after, in a medium with normal Krebs solution, the recuperation was assessed. Results are expressed in milligrams of contraction; each column represents the mean, with vertical lines showing SEM of 5 experiments. Significant differences from control values where *P < 0.05 (Student’s unpaired t-test).

 
The preincubation of the preparations with nifedipine (1 µM) or with {omega}-conotoxin GVIA (0.1 µM; antagonists of L- and N-type of Ca2+ channels, respectively), 20 minutes prior, did not result in any significant change in the contractile response induced by SP (Figs. 4A and 4B ). However, nickel (1 mM, a nonselective blocker of Ca2+ channels), preincubated with the preparations 20 minutes beforehand, significantly inhibited (69.49% ± 7.63%) SP-induced contraction in the isolated rat iris (P < 0.05; Fig. 4C ).



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  		Figure 4. Cumulative log concentration-effect curves for SP obtained in the isolated rat iris in the absence or presence of nifedipine (1 µM; A), {omega}-conotoxin (1 µM; B), and nickel hydrate (1 mM; C). Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing SEM of 5 to 6 experiments. Significant differences from control values where *P < 0.05 (Student’s unpaired t-test).

 
Influence of Protein Kinase C and Tyrosine Kinase on SP-Mediated Contraction
Preincubation of the preparations with the protein kinase activator phorbol ester (10 µM), for 30 minutes, did not result in any variation in the tonus of the preparation nor did it interfere significantly with the contraction induced by SP in the rat iris (Fig. 5A ). Staurosporine (1 µM), a protein kinase C inhibitor, added to the preparations 30 minutes prior, produced a small but significant shift to the right without changing the Emax for SP (Fig. 5B) . In contrast, the preincubation of the preparations with the tyrosine kinase inhibitor genistein (1 µM) had no significant effect on SP-mediated contraction in the rat iris (Fig. 5C) .



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  		Figure 5. Cumulative log concentration-effect curves for SP in the isolated rat iris in the absence or presence of phorbol (10 µM; A), staurosporine (1 µM; B), and genistein (1 µM; C). Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing SEM of 4 to 5 experiments. Significant differences from control values where *P < 0.05 (Student’s unpaired t-test).

 
Influence of NO and CGRP on SP-Mediated Contraction
L-NAME (10 µM), an inhibitor of NO synthase (NOS), when added to the preparations 20 minutes prior, produced a small but long-lasting contraction in the rat iris (33.31 ± 8.50 mg of tension). However, L-NAME (0.10–1000 µM) did not significantly affect SP-mediated contraction in the rat iris (Fig. 6) .



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  		Figure 6. (A) Contractions induced by SP (1 µM) and L-NAME (10 µM) in the isolated rat iris and (B) effects of L-NAME (0.1–100 µM) on the contractile response induced by SP (100 nM). Results are expressed in milligrams of contraction; each column represents the mean, with vertical lines showing SEM of 5 experiments.

 
The cumulative addition of CGRP to the preparations caused a small though concentration-dependent contraction (Fig. 7A ). In the presence of CGRP (30 µM), the contractile CRC for SP was significantly enhanced (20.53% ± 6.32%), but its EC50 was not significantly affected (Fig. 7A) . The preincubation of the preparations with CGRP (8-37) fragment (an antagonist of CGRP) did not significantly affect the SP-induced contraction in the rat iris (Fig. 7B) .



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  		Figure 7. (A) Cumulative log CRCs for SP, CGRP, and SP plus CGRP (30 µM) and (B) log concentration-effect curves for SP in the absence or presence of CGRP (8-37) in isolated rat iris. Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing the SEM of 5 experiments.

 
Concentration-Response Relationships for Selective Tachykinin Agonists
Cumulative addition to the bath of the natural selective tachykinin agonists NKA or NKB (0.001–1000 nM) resulted in a concentration-dependent contraction of the rat iris. The potency (EC50) and Emax for NKA and NKB did not differ significantly from that of SP (3.49; 0.89–13.77 versus 7.03, 1.94–25.52 and 3.89, 0.91–16.42 nM, respectively Fig. 8A ). The synthetic selective NK3 and NK2 tachykinin agonists senktide (0.001–1000 nM) and [ß-ala] NKA (0.1–1000 {eta}M) also produced concentration-dependent contraction in the rat iris. The [ß-ala] NKA had the same potency as SP (0.23, 0.12–0.43; 34.62, 22.24–53.89; and 27.47, 0.54–137.77 nM, respectively; Fig. 8B ). At the EC50 level, senktide was approximately 150-fold more potent than the NK2 receptor selective agonist [ß-ala] NKA.4 5 6 7 8 9 10 However, the Emax produced by the three peptides did not differ significantly (Fig. 8) . In marked contrast, the selective NK1 agonist SP methyl ester (0.1–1000 nM) caused a very weak contraction in the rat iris (Emax 9.36 ± 1.57 mg; Fig. 8B ).



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  		Figure 8. Cumulative log concentration-effect curves in isolated rat iris for natural tachykinin agonists ([{circ}], SP; [{blacksquare}], NKA; and [{blacktriangleup}], NKB; A) and for selective synthetic tachykinin agonists ([{circ}], SP, control; [•], senkitide; [{blacktriangleup}], SP methyl ester; [{blacksquare}], [ß-ala] NKA; B). Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing the SEM of 5 to 6 experiments.

 
Actions of Specific Tachykinin Receptor Antagonists on SP-Mediated Contraction
The NK3 antagonist SR 142801 (1–10 nM) antagonized the contractile responses to SP, resulting in concentration-dependent rightward shifts in the agonist curves (Fig. 9A ). The mean pA2 was calculated to be 9.37 (±0.52), with a slope of 0.70 [0.32;1.08] (n = 4) and a correlation rate of 0.96 (Fig. 9C) . The preincubation with SR 48968 (10–100 nM), a NK2 antagonist, 30 minutes before, also resulted in a concentration-dependent shift to the right of SP-mediated contraction without changing the maximal response (Fig. 9B) . The estimated pA2 value was 7.97 (±0.15), with a slope of 1.02 [0.59;1.47] and a correlation rate of 0.77 (Fig. 9C) .



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  		Figure 9. (A) Cumulative log concentration-effect curves for SP in the isolated rat iris obtained in the absence or presence of different concentrations of SR 142801. (B) Cumulative log concentration-effect curves for SP in the isolated rat iris obtained in the absence or presence of different concentrations of SR 48968. Results are expressed in milligrams of contraction; each point represents the mean, with vertical lines showing the SEM of 4 to 5 experiments. (C) Schilds plots for SR 142801 and SR 48968 as antagonist of SP in isolated rat iris.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the first study showing that SP induces a powerful and concentration-dependent contraction in the isolated rat iris, being approximately 40-fold more potent than the cholinergic agent carbachol but having a similar efficacy. Our findings further extend the previous study of Banno et al.,16 where, when field stimulation is used, SP might be the element mainly responsible for the non-adrenergic, noncholinergic response in the rat iris. Interestingly, our findings also show that, in contrast to many other tissues,18 19 20 the contractile response induced by SP in the rat iris seems to be resistant to iris proteases, evident from the finding that preincubation of the preparations with three known proteases inhibitors (captopril, phospharamidom, and thiorphan) did not significantly affect SP-mediated contraction. However, the role played by other peptidases cannot be completely discarded, because the serine protease inhibitor diisopropyl fluorophosphate has been reported to inhibit the degradation of SP in the aqueous humor of rabbits and dogs.21

There is compelling evidence indicating that in some tissues SP can induce neural release of acetylcholine,22 23 an effect that has been demonstrated to be sensitive to atropine, a cholinergic antagonist. Our results have also demonstrated that the contraction induced by SP in the isolated rat iris is in great part mediated by neural release of acetylcholine, as atropine greatly shifted SP-mediated contraction to the right. Further evidence for an indirect neural action of SP-mediated contraction in the rat iris has been demonstrated by the use of TTX, which also markedly displaced SP-mediated contraction to the right, indicating that neuronal release is probably involved. However, there are no descriptions concerning the involvement of the cholinergic system in SP contraction of the iris in other animal species. Furthermore, our results also show that contraction induced by SP in the rat iris is probably not related to the activation and/or release of histamine or adrenergic amines, because pyrilamine and guanethidine at concentrations known to block histamine and the release of adrenergic amines had no significant effect on SP response in the rat iris. Also, the involvement of metabolites of the cyclo-oxygenase pathway seems improbable because indomethacin failed to affect SP-mediated contraction.

In ocular tissues, in most species studied, SP has been identified in sensory nerves originating from trigeminal ganglion in association with CGRP.24 In the rat, about 20% of trigeminal ganglion cells are immunoreactive to SP, whereas approximately 40% are immunoreactive to CGRP.25 In other species, such as rabbits, both SP and CGRP are considered to coexist in sensory nerve fibers,10 and when released after noxious stimuli26 they are associated with the increase of intraocular pressure breakdown of blood-aqueous humor, but have little effect on the pupil diameter of the rabbit.27 Results of the present study have also demonstrated that CGRP induces a small but concentration-dependent contraction in the rat isolated iris, suggesting that it might exert a physiological role in these preparations. Interestingly, when preparations were preincubated with CGRP, there was a significant increased in SP-induced contraction. However, there is no evidence to support the release of CGRP in the response induced by exogenous SP, because the addition of 8-37 CGRP fragment, a selective antagonist of CGRP, at a concentration known to block SP response, had no significant effect on SP-induced contraction in the rat iris. It has been reported that in the rabbit iris dilator muscle, CGRP appears to participate in SP-induced relaxation.28

Pharmacological and biochemical studies have demonstrated that responses elicited by SP and related tachykinins in vascular and nonvascular smooth muscles depend on the activation of phospholipase C, with consequent production of inositol(1,4,5)-triphosphates that in turn induces intracellular Ca+2 release and muscle contraction.29 30 31 32 In the isolated rat iris, the contractile response elicited by SP appears to rely largely on Ca2+ influx from extracellular sources, because omission of Ca2+ from the medium in the presence of EGTA (1 mM) almost abolished (approximately 80%) SP-mediated contraction. Normally, the Ca2+ ions enter into cells through the voltage-dependent channels, which are opened on depolarization of the membrane, mainly through the L and N types.33 34 In the isolated rat iris, the Ca2+ influx induced by SP seems unlikely to be associated with the activation of L and N types of Ca2+ channels, because nifedipine and {omega}-conotoxin were largely ineffective in antagonizing SP response. However, nickel, a nonselective Ca2+ channel blocker, consistently antagonized SP-mediated contraction in the rat iris. Thus, the use of selective antagonists of T, P, or R channels is required to prove which type of Ca2+ channel is involved in SP-mediated contraction in the rat iris.

The results of the present study also suggest that activation of the protein kinase C mechanism is not involved in the contraction induced by SP in the rat iris, because staurosporine, a potent though nonselective antagonist of protein kinase C,35 caused only a marginal inhibition of SP-induced contraction. A further piece of evidence that supports this assumption is the fact that the addition of phorbol ester to the preparations, a specific activator of protein kinase C,30 36 37 failed to elicit any contraction in the rat iris, nor did it change the contractile response induced by SP. Furthermore, genistein, a tyrosine kinase inhibitor, also failed to alter the response induced by SP, suggesting that this pathway is not involved in its action.

Recently, it has been demonstrated that NO serves as a mediator for nonadrenergic, noncholinergic nerves in respiratory, digestive, genitourinary, and vascular systems.38 The participation of NO in the iris contraction has been suggested by the presence of NOS activity in the rabbit iris sphincter.39 The contraction induced by tachykinin in the rabbit iris seems not to have been mediated by NO.40 In the present study, L-NAME, an inhibitor of NO formation, induced a sustained tonic contraction of the isolated rat iris, although it did not change the contractile response to SP. These findings suggest that NO could have a physiological role in the basal muscle tonus maintenance of the rat iris, but NO itself has apparently no major influence on SP-induced contractile response in this preparation.

To the best of our knowledge, the subtype(s) of tachykinergic receptors involved in SP-induced contraction of the rat iris have never been described. In regard to rabbits, for a long time, there has been a controversy about the subtype of receptor involved. In the past some authors believed that contraction induced by SP in the rabbit iris was due to activation of NK1 receptors alone41 42 or in association with NK3 receptors,43 whereas others supported the involvement of NK2 receptor.40 More recently, it has been shown that the rabbit iris contraction depends on the sole activation of NK3 receptors.14 44 Our results clearly show for the first time that all the natural tachykinin agonists (NKA, NKB, and SP) cause concentration-dependent contraction in the rat iris, without any significant difference in the potency or maximal developed tension. However, when synthetic agonists were tested, [ß-ala] NKA (an NK2 agonist) and senkitide (an NK3 agonist) also induced concentration-dependent contractile response in rat iris, the NK3 selective agonist senktide being approximately 150 times more potent than the NK2 selective agonist [ß-ala] NKA, but with similar maximal response. On the other hand, SP methyl ester, an NK1 selective agonist, induced a small contractile response. These findings strongly suggest the possible coexistence of at least two populations of tachykinergic receptors in the rat iris (NK2 and NK3).

To explore further the receptor subtypes that mediate SP-induced contraction in the rat iris, we carried out an experiment using specific tachykinin antagonists. SR 48968 and SR 142801, the potent nonpeptide NK3 and NK2 antagonists,7 31 caused concentration-dependent displacement to the right of the curves for SP with no change in the maximal response, furnishing the pA2 values of 9.37 and 7.97 and slopes of 0.70 and 1.02, respectively. Such findings further suggest that SP-induced contraction in the rat iris is mediated jointly by activation of NK2 and NK3 receptors. The affinity estimated for SR 142801 in antagonizing SP contraction in the rat iris was slightly higher than that shown against senktide in rabbit iris (pA2 8.9)14 but was comparable to that obtained in the guinea pig ileum and in human NK3 receptors expressed in Chinese hamster ovary cells (pKB, 8.98–9.27; pKi, 9.4–9.68, respectively),45 46 it was certainly different from the affinity for NK3 receptors found in the rat (pKB, 7.49).46 The existence of differences among species in relation to the affinity to NK3 receptors has already been shown. Usually, the rat and rabbit show similar responses, but the NK3 antagonist SR 142801 exhibits higher affinity in humans and in guinea pigs. Thus, the NK3 affinity found in the rat iris seems to be quite similar to those reported for human and guinea pig tissues.

SR 48968 (a nonpeptide, potent NK2 antagonist whose recent studies have demonstrated a limited but sizable binding affinity for NK3 receptors in the guinea pig and rat cerebral cortices47 ) also caused a concentration-dependent displacement to the right of the curves elicited by SP, with a pA2 value of 7.97 and a slope of 1.02. In rabbit vena cava, SR 48968 shows some weak inhibitory activity against SP (pA2 6.08), and a pKB of 7.63 in guinea pig isolated common bile duct tissues.18 When assessed against selective NK2 agonists, SR 48968 has higher pA2 values (between 9.6 and 10.3) in guinea pig, rabbit, and human receptors48 than in tissues from hamsters and rats (pA2 values between 7.5 and 8.7).31 In rabbit iris sphincter muscle, SR 48968 antagonized senktide-induced contraction, furnishing a pA2 value of 6.1.14 Results of the present study revealed an intermediary affinity for SR 48968 that was higher than that reported by other authors in studies on rat tissues.31 Because clearly the rat iris shows two tachykinin receptors (NK2 and NK3), a definitive pharmacological characterization of such receptors depends on the use of more selective agonists and antagonists.

In summary, the results of the present study have clearly demonstrated that SP produces a concentration-dependent contractile response in isolated rat iris, being approximately 40 times more potent than carbachol. At least in part, the SP-mediated contraction was mediated by the endogenous release of acetylcholine and depended on nerve-mediated action potential sensitive to TTX. In marked contrast to the reported response to SP in other tissues, in the rat iris its action relies largely on external Ca2+ channel influx by a mechanism that seems to depend on the activation of Ca2+ channels, different from the types L or N, and that seems not to be associated with activation of either protein kinase C or tyrosine kinase–mediated mechanisms. The CGRP, when used alone, caused a small contraction of rat iris and an additive response together with SP, an action that could suggest the coparticipation of these peptides after release by a noxious stimulus. Finally, our results using both selective agonists and competitive antagonists strongly suggest that SP-mediated contraction in the rat iris is mediated by the activation of NK2 and NK3 receptors.


   Footnotes
 
Supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Brazil. MAD and RVA are undergraduate medical students supported by fellowships through CNPq.

Submitted for publication June 15, 1999; revised November 24, 1999; accepted December 24, 1999.

Commercial relationships policy: N.

Corresponding author: João Batista Calixto, Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Rua Ferrreira Lima, 82, CEP-88015-420, Florianópolis, SC, Brazil. calixto{at}farmaco.ufsc.br


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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