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Secretion and serotonin release in the isolated rat lacrimal gland: the effects of substance P and calcitonin gene-related peptide
Journal of the
ELSEVIER
Journal of the Autonomic Nervous System 61 (1996) 37-42
Autonomic Nervous System
Secretion and serotonin release in the isolated rat lacrimal gland: the effects of substance P and calcitonin gene-related peptide Ruth M. Williams a.b, Lorenzo Bauce a, Robert W. Lea h, Jaipaul Singh b, Keith A. Sharkey a,* a Neuroscience Research Group, Department of Medical Physiology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada b Cell Communication Group, Department of Applied Biology, University of Central Lancashire, Preston PR1 2HE, UK Received 1 September 1995; revised 17 January 1996; accepted 27 January 1996
Abstract A close anatomical relationship between nerves containing substance P and caicitonin gene-related peptide (CGRP) and mast cells containing serotonin has been demonstrated in the rat lacrimal gland. This study investigates the potential for peptidergic regulation of lacrimal mast cells by examining the actions of substance P, CGRP and serotonin on protein and peroxidase secretion from isolated lacrimal segments and on substance P and CGRP to release serotonin from the lacrimal mast cells. Substance P, CGRP and serotonin evoked marked increases in total protein and peroxidase from the lacrimal. Sodium cromoglycate, a mast cell stabilizer, significantly reduced or blocked the secretory responses elicited by these agonists. Chromatographic analysis using electrochemical detection revealed Ihat substance P, but not CGRP, augmented the release of serotonin from the gland. The substance P evoked peroxidase secretion and serotonin release was blocked by CGRP and by sodium cromoglycate. These results support a role for mast cells in the regulation of lacrimal secretion and suggest a novel regulatory interaction between substance P and CGRP in the control of lacrimal function through a meuro-immune interaction. Keywords: Sodium cromoglycate; Mast cells; Substance P; 5-HT; CGRP; Neuroimmunology
1. Introduction The secretion of tears by the exorbital lacrimal gland is primarily under the control of the autonomic nervous system [2,6,17]. Secretion elicited by electrical field stimulation of isolated lacrimal glands was shown to be in part resistant to combined autonomic blockade [16], suggesting a non-adrenergic, non-cholinergic (NANC) innervation of the gland. These NANC nerves were believed to be peptidergic and immunohistochemical studies have since revealed an extensive peptidergic innervation of the lacrimal in several species, including man [9,22-24,33,35,39]. The precise physiological role of many of these peptides has yet to be defined, but it is suggested that some may function as secretomotor transmitters in the regulation of lacrimation [9,10,18].
* Corresponding author. Tel.: + 1 403 2204601; fax: + 1 403 2838731; e-mail: ksharkey @acs.ucalgary.ca
In a recent study of the rat lacrimal it was demonstrated that nerves containing substance P and calcitonin gene-related peptide (CGRP) were closely associated with mast cells in the gland [39]. Elsewhere, such as the skin and GI tract, a close association between mast cells and peptidergic nerves has been reported [34,36]. These peptides have previously been studied for their potential role in the modulation of mast cell secretion and more generally in the modulation of immune cell activity [30,31,34,37]. Mast cells have a secretory capacity. In mouse and rat, the released mediators include the biogenic amines histamine and serotonin (5-HT), which have pronounced biological effects that include exocrine secretion from glands [18,8,13,25,29,37]. Classically, the release of mast cell mediators is induced by an antigen-antibody (IgE) reaction on the mast cell membrane [1,13]. Recent evidence suggests that neural mechanisms are also involved in mast cell stimulation. It is now apparent that neuropeptides can modulate some immune responses directly (see [37] for a review) and several peptides, notably substance P, can modulate mast cell mediator release [1,12,13,37]. Since
0165-1838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII SO 165-1 838(96)0003 1-8
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R.M. Williams et al. / Journal of the Autonomic' Nervous System 61 (1996) 37-42
mast cell mediators and neurotransmitters can potentially elicit lacrimal secretion and since there is evidence for a functional association between nerves and mast cells in the skin [12,26,34], we sought to test the hypothesis that there is a functional relationship between nerves and mast cells in the rat lacrimal gland. To do this we examined the action of substance P and CGRP in eliciting secretion in the isolated lacrimal gland in the presence and absence of the mast cell membrane stabilizer sodium cromoglycate. Since we had evidence that 5-HT was present in mast cells in the rat lacrimal [39] we also studied the effects of 5-HT on secretion. Finally, we measured the effects of the substance P and CGRP on 5-HT release from isolated glands. A preliminary account of part of this work has been previously published [38].
2. Materials and methods 2.1. Animals
All the procedures described below were approved by institutional (Canada) and national (UK) Animal Care Committees. Experiments were performed on adult male Sprague-Dawley rats (200-250 g; Charles River, Quebec, Canada; Charles River, Sussex, UK). Rats were housed under constant photoperiod (14 h light, 10 h dark) and temperature (20°C) and were allowed food and water ad libitum. 2.2. Tissue preparation
Rats were killed by cervical dislocation and exsanguination. Lacrimal glands were quickly excised and placed in a modified oxygenated (95% 0 e / 5 % CO 2) Krebs-Henseleit (KH) solution of the following composition (mM): NaCI, 118; KC1, 3.7; CaC12, 2.56; MgCI 2, 1.2; NaHCO 3, 25; KH2PO 4, 2.2; D-glucose, 10; pH 7.3-7.4. The connective tissue capsule was carefully and completely removed from each gland, which was then sliced into small segments (5-10 mg). In each experiment about 25% of a gland was used (30-35 mg). Lacrimal segments were washed (5 times) with KH solution and placed in 5 ml plastic tubes containing either KH solution alone (control) or KH solution containing different concentrations of test substances. Segments were incubated for a basal period of 20 min at 37°C in a shaking water bath, then KH was removed and replaced with the same volume of KH containing the test compound. Tissues were incubated for a further 20 min. At the end of the final incubation period, samples were collected and analyzed as described below. In studies using sodium cromoglycate (a gift from Fisons Pharmaceuticals, UK), tissues were preincubated for an additional 15 min prior to the addition of agonists which were incubated for 20 min as previously described. In each case, tissues were tested with only a single concentration of agonist in the
presence or absence of sodium cromoglycate. At the end of each experiment tissues were removed, blotted dry and weighed. 2.3. Peroxidase assay
Peroxidase activity was measured as a marker of acinar cell secretion using a modification of the method of Herzog and Fahimi [14]. Briefly, 200 I~1 of the incubation medium was added to 3,3'- diaminobenzidine (5 raM) in sodium phosphate buffer (0.1 M, pH 7.4). Hydrogen peroxide was added and the contents mixed. The optical density was measured immediately at 460 nm using a Visi spectrophotometer (Pharmacia, UK). Absorbance was measured over 3 min and the slope of the measurements used to determine peroxidase activity using a peroxidase (Sigma) standard and KH solution as a blank. All values are expressed as ng ml-1 100 rag-J tissue. 2.4. Protein assay
The protein content of the samples was determined spectrophotometrically using a modification of the Bradford assay [3], described by Bromberg and Welch [7]. Samples of incubation medium were added to the protein reagent and mixed. Optical density was recorded at 595 nm using a Visi spectrophotometer (Pharmacia, UK). Absorbance was measured after 30 min using KH solution as a blank and bovine serum albumin (Sigma) as a standard. All assays were performed in duplicate. Concentrations were determined from standard curves constructed for each series of experiments. Protein secretion was expressed as m g m l J 100mg ~ tissue. 2.5. Serotonin release
Perchloric acid (100 pA) was added to a sample of the incubation medium (900 Ixl) to precipitate protein. The mixture was shaken and left for 10 min on ice. After centrifugation (10,000 × g for 1 min), the supernatant was analyzed for 5-HT by high performance liquid chromatography (HPLC) using an electrochemical detection system. Known concentrations of 5-HT (Sigma) were used as standards. Samples were separated on a Beckman Ultrasphere Octadecylsilica column (4.5 × 50 mm, 5 Ixm) using a mobile phase consisting of 0.2 M acetic acid, 0.05 M sodium acetate, 100 m g / l EDTA, 100 mg/1 sodium dodecylsulphate and 25% methanol ( v / v ) at a flow rate of 0.5 m l / m i n at 40°C. Detection was by means of a BAS LC4B amperometric detector with a glassy carbon electrode operating at +0.60 V. The signal was digitized, stored and quantitated using the external standard method using a Waters Maxima data acquisition system. Results are expressed as pmol 100 mg -~ tissue. In order to assess the total 5-HT content of the lacrimal, glands were homoge-
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R.M. Williamset al. / Journal of the Autonomic Nervous System 61 (1996) 37-42
a i z e d in perchloric acid and the supernatant analyzed as described above.
A. A 0
Ji"
2.6. Statistical analysis
!-
Data are presented as mean ___ standard error o f the mean (SEM) from 5 to 8 experiments using glands from different animals. Data were compared using A N O V A followed by N e w m a n - K e u l s post hoc test, or unpaired Student's t-tests as appropriate. Differences between values were considered significant at P < 0.05.
3.1. Lacrimal secretion
Two measures o f lacrimal secretion were used in the Wesent study: peroxidase release and total protein release. In all cases, parallel effects were observed with either assay, where comparable experiments were performed. Basal secretion of protein (measured in one series of ~ltperiments) was 1.46-t-0.1 mg ml - I 100 mg -= tissue ( n = 6) and basal peroxidase secretion 32.8 + 4.4 ng m l - 1 190 mg - ] tissue (n = 24).
2-
c
"
-, ......... 10-9
, ........ . ........ . 10-8 1 0 -), 10-6
Substance P a.
3. Results
4-
. . . . . . . .
i
.
.
10-~J
(M)
A
E; '6,:'.
substance P
substance ÷ Na
P
cromoglycate
Fig. 1. (A) The effect of varying concentrations of substance P on protein release from isolated segments of lacrimal gland. Substance P caused a concentration-dependent increase in protein release (basal subtracted) at concentrations above 10 -9 M, with a maximal effect at about 10 - 6 M. (B) The effect of sodium cromoglycate (10 -4 M) on substance P (10 - 6 M) induced protein release. Sodium cromoglycate virtually abolished protein release (*, P < 0.01) evoked by substance P. Each point is a mean of 5-8 responses from different glands.
3,2. Substance P Substance P evoked a concentration-dependent protein release with a maximal effect at 10 -6 M and an ECs0 of 2 X 10 -8 M (Fig. 1). Similarly, peroxidase release was enhanced over a similar concentration range and EC50 (7 × 10 -8 M)(Fig. 2). In both cases the response to substance P was blocked by prior addition of the mast cell stabilizer sodium cromoglycate (10 -4 M). This concentration of sodium cromoglycate had no significant effect on basal secretion of either protein or peroxidase (data not shown).
3.4• Serotonin
Since 5-HT has been reported to be a secretagogue in other exocrine glands and is stored in mast cells of the lacrimal gland we examined the effects of 5-HT on protein and peroxidase secretion. 5 - H T caused a concentrationdependent peroxidase release at a somewhat reduced potency compared to either substance P or C G R P (EC50 5 X 10 -7 M) (Fig. 4). Protein secretion was also enhanced
100-
3,3. CGRP
C G R P evoked protein release (at the concentration tested (10 -7 M)), and at this and higher concentrations a peroxidase secretion, similar in magnitude to that evoked by equimolar concentrations o f substance P (Fig. 3). The BC50 of the response (9 x 10 -8 M) was not significantly different from that of substance P. The effects of combinillg substance P and C G R P were examined for peroxidase secretion. It was found that at the two concentrations tested, there was a marked attenuation in the effect of C G R P in the presence of substance P (Fig. 3). The effect o f sodium cromoglycate was examined only for protein secretion. In this case, there was no effect o f the mast cell stabilizer ( P = 0.17) on the C G R P evoked protein secretion.
J!
o Krebs
50-
•
Substance Substance
P P +
Na Cromoglycate ' ,"o-; "~o-; "i'o-; "~'o-' 'i~-; 'i~-' Substance P [M]
Fig. 2. The effect of varying concentrations of substance P (filled squares) on peroxidase release from isolated segments of lacrimal gland. Substance P caused a concentration-dependent increase in peroxidase release above basal (basal shown by the open circle) at concentrations above 10-s M, with a maximal effect at about 5 X 10 -6 M. The effect of sodium cromoglycate (10 -4 M) on substance P-induced peroxidase release (filled triangles). Sodium cromoglycate abolished peroxidase release (*, P < 0.05, ANOVA) evoked by substance P. Each point is a mean of 5-8 responses from different glands.
R.M. Williams et al. / Journal c)[the Autonomic Nervous System 61 (1996) 37-42
40 A.
100-
75"
50"
• CGRP
o ~ E-
~g
by 5-HT at the concentration tested (10 _5 M, which caused maximal peroxidase release). This effect was substantially reduced by pretreatment of the tissue by sodium cromoglycate, suggesting that 5-HT was acting to degranulate mast cells to cause its effect.
• CGRP + substance p
25"
3.5. Serotonin release . . . . . . . . . . . . . . . .
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0
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. . . . . . .
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. . . . . . . . . . .
10.7
10.6
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B.
CGRP
CGRP
+ Na cromoglycate
Fig. 3. (A) The effect of varying concentrations of CGRP (filled squares) on peroxidase release from isolated segments of lacrimal gland. CGRP caused a concentration-dependent increase in peroxidase release at concentrations above 10 -8 M. Equimolar mixtures of CGRP and substance P (filled triangles) blocked the peroxidase release (*, P < 0.05). (B) The effect of sodium cromoglycate (10 -4 M) on CGRP (10 7 M)-induced protein release (basal subtracted). Sodium cromoglycate had no effect on protein release evoked by CGRP. Each point is a mean of 5 - 8 responses from different glands.
A.
Mean basal release of 5-HT was 4.3 _+ 0.4 pmol 100 mg -t tissue (n = 15). Chromatographic analysis of the effluent samples in response to substance P demonstrated that substance P evoked a two-fold increase in 5 - HT release at concentrations above 10 8 M (10 8 M, 9.9 + 0.7 pmol 100mg ~ tissue; 10 6 M, 9 . 2 _ + 0 . 5 p m o l l 0 0 m g - t tissue). Lower concentrations (10 1 0 - 1 0 - 9 M ) failed to stimulate 5-HT release. Pretreatment of the tissue with sodium cromoglycate (10 4 M) significantly reduced, to slightly below basal levels, 5-HT release in response to 10 - 6 M substance P (3.0 + 0.1 pmol 100 mg -~ tissue). CGRP was without effect on 5-HT release at concentrations of 10 1 0 - 1 0 - 6 M and indeed at the highest concentration (10 6 M) levels were significantly below basal (2.5 _+ 0.4 pmol 100 m g - ] tissue, P < 0.05). As for peroxidase secretion, addition of CGRP blocked the effects of substance P ( 1 0 - 6 M substance P + 10 6 M CGRP; 3.45 _+ 0.4 pmol 100 mg l). The total content of 5-HT in the rat lacrimal gland was 9 5 + 4 . 5 pmol 100 mg a tissue ( n = 5 ) . Based on this data it appears that substance P maximally stimulated the release of about 9% of the total glandular 5-HT content.
15o.
4. Discussion x ~. o-~
o
io-;*'
1o-'
1o"
to-'
1o"
lo-'
1o-'
5-HT (M) B.
|
5-HT
5-HT
Na c r o ~ g l y c a t e
Fig. 4. (A) The effect of varying concentrations of 5-HT (filled squares) on peroxidase release from isolated segments of lacrimal gland. 5-HT caused a concentration-dependent increase in peroxidase release at concentrations above 10 - s M, with a maximal effect at about 10 5 M. (B) The effect of sodium cromoglycate (10 -4 M) on 5 - H T (10 5 M)-induced protein release (basal subtracted). Sodium cromoglycate significantly reduced ( *, P < 0.05) protein release evoked by 5-HT. Each point is a mean of 5 - 8 responses from different glands.
Exogenous application of substance P to isolated lacrimal segments resulted in a concentration-dependent peroxidase and protein secretion. This secretory response could be almost totally abolished by pretreatment of the tissue with the mast cell stabilizer sodium cromoglycate. Application of CGRP and 5-HT to isolated lacrimal segments evoked a concentration-dependent peroxidase release and significant protein secretion. Whereas secretion elicited by 5-HT could be attenuated by sodium cromoglycate, that evoked by CGRP was not blocked. These findings implicate the mast cell as an intermediary in the regulation of lacrimation by neuropeptides. Furthermore they suggest a role for mast cell mediators in tear secretion and their potential modulation by nerves containing substance P and CGRP. These findings extend the anatomical observations previously presented [39], demonstrating a close relationship between 5-HT-containing mast cells and nerve fibers containing substance P and CGRP in the rat lacrimal gland. Substance P has previously been demonstrated to degranulate isolated mast cells obtained from different sources
R.M. Williams et al. / Journal of the Autonomic Nervous System 61 (1996) 37-42
representing the two heterogeneous classes of mast cell described in rats (connective tissue [peritoneal] type mast cells and mucosal mast cells) [11,13,37]. In addition, studies in vivo show that substance P can degranulate mast cells in the skin [26]. The mechanism of action of substance P has previously been described as a receptor-independent pathway [27], which may in part explain the lack of concentration-responsiveness of 5-HT release. More te.cently, however, this idea has been challenged suggesting that substance P can act via NK1 and NK2 neurokinin receptors on mast cells [20,21]. CGRP is considerably less effective as a mast cell ~,cretagogue when compared to substance P [12,31]. However, previous studies have shown that substance P can attenuate the action of CGRP through the release of mast Gell proteases [4,5]. In this study not only was CGRP unable to stimulate 5-HT release, its agonist action on protein release was unaffected by a mast cell stabilizer. Xowever, CGRP was apparently able to interact with substance P to effectively prevent the action of substance P. The mechanism whereby CGRP exerts its inhibitory actions is not apparent from this study, but these results ~uggest a possible new regulatory interaction between these two peptides in lacrimal physiology. In the lacrimal gland the role of 5-HT has not previously been examined. Based on immunohistochemical ob~rvations the sole source of 5-HT in the gland is mast qells [39]. Consistent with this observation, the mast cell ~labilizer sodium cromoglycate effectively suppressed the 5-HT release induced by substance P and reduced protein release induced by substance P and 5-HT, but not CGRP. This suggests that the protein secretory effect of substance P and 5-HT is indirectly mediated by mast cells, whose released mediators give rise to lacrimal secretion. Why the ~ f e c t of substance P on 5-HT release is not ooncentration-dependent is unclear. As mentioned above, it may degranulate mast cells in a receptor-independent manaer [27]. Also, the apparent lack of concentration-responsiveness might be explained by rapid uptake and degradation mechanisms for 5-HT that exist and that mast cell degranulation may not be an entirely graded phenlomenon. The concentration-dependency of the secretion data may then be due to actions of the peptide on mast cells and the net effect of the mast cell mediators being a ~lraded response through activation of receptors on the acinar cells of the lacrimal. Which mast cell mediator gives rise to secretion in tmknown, but histamine and 5-HT are recognized in other e~xocrine glands as secretagogues [8,25,29]. The capacity of 5-HT to degranulate mast cells has previously been examreed [19]. Serotonin was found not to degranulate rat ~ritoneal mast cells in vitro; however, the staining characteristics of lacrimal mast cells are not entirely consistent with them being of this type [39]; hence interpretation of the effects of 5-HT on lacrimal mast cells requires further investigation.
41
The mast cell-nerve relationship suggested by the results of the present study is worthy of additional study, since several important questions remain unanswered. Firstly, is this an obligatory functional unit, such that the release of substance P always causes mast cell activation? If that is the case, and substance P is in primary afferent nerves, then is there a neurogenic lacrimation analogous to neurogenic inflammation in the skin [15]? In this context, sensory reflex lacrimation stimulated by a substance P analogue (eledoisin) has been reported in the rabbit [28], but the same compound applied intraarterially to the lacrimal failed to elicit lacrimal secretion [10]. Conversely, is there bidirectional communication and can mast cells activate nerves in the gland? In either case answers to these questions are important in the context of allergy, as tear production may be affected in situations where mast cell degranulation occurs, e.g. in certain allergies. Previous studies in the lacrimal gland have suggested an endocrine, neural and immune control of the release of secretory component from isolated acinar cells [18]. Secretory component is the lgA receptor and functions to transfer IgA into tears. In this study substance P and CGRP were found not to stimulate (or inhibit) secretory component, whereas vasoactive intestinal polypeptide stimulated its release and a cholinergic agonist inhibited its release [18]. Certain cytokines also stimulated the release of secretory component (interleukin 1 and tumor necrosis factor). Since cytokines are released from mast cells [1,13], it is possible that under conditions where mast cells are stimulated (such as the present study) secretory component is also released in association with other proteins to effect the protective actions that they serve in the normal physiology of the eye. In conclusion, the present study demonstrates that neuro-immune interactions are probably a normal part of lacrimal secretory physiology or pathophysiology, as is being increasingly recognized as a fundamental feature of most autonomically innervated tissues [32,36]. The added complexity endows the autonomic nervous system with additional roles that suggests it functions in a more integrative manner than was previously suspected. Finally, a better understanding of the interactions between the nervous and immune systems in the lacrimal gland may offer novel avenues for therapeutic intervention in diseases such as keratoconjunctivitis sicca and other diseases of aqueous tear film deficiency.
Acknowledgements This work was supported by the Medical Research Council of Canada (grant to KAS), The British Council Academic Links Scheme (to K.A.S. and J.S.), and the Wellcome Trust (grant to J.S.). K.A. Sharkey is an Alberta Heritage Foundation for Medical Research Scholar. R.M. Williams received a Studentship from the Science and
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R.M. Williams et al. / Journal of the Autonomic Nervous System 61 (1996) 37-42
Engineering Research Council. We thank Fisons Pharmaceuticals (UK) for generously providing the sodium crom o g l y c a t e u s e d in this s t u d y .
References [1] Befus, A.D., Bienenstock, J. and Denburg, J.A., Mast cell differentiation and heterogeneity, Raven Press, New York, 1986. [2] Botelho, S.Y., Hisadad, M. and Fuenmayor, N., Functional innervation of the lacrimal gland in the cat, Arch. Ophthalmol., 76 (1966) 581-588. [3] Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of proteindye binding, Analyt. Biochem., 72 (1973) 248-254. [4] Brain, S.D. and Williams, T.J., Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin, Br. J. Pharmacol., 97 (1989) 77-82. [5] Brain, S.D. and Williams, T.J., Substance P regulates the vasodilator activity of calcitonin gene-related peptide, Nature, 335 (1988) 73-75. [6] Bromberg, B.B., Autonomic control of protein secretion, Invest. Opthalmol. Vis. Sci., 20 (1981) 110-116. [7] Bromberg, B.B. and Welch, M.H., Lacrimal protein secretion: comparison of young and aged rats, Exp. Eye Res., 40 (1985) 313-320. [8] Chernick, W., Bobyock, E. and Bradford, P., 5-Hydroxytryptamine modulation of rat parotid salivary gland secretion, J. Dent. Res., 68 (1989) 59-63. [9] Dartt, D.A., Baker, A.K., Vaillant, C. and Rose, P.E., Vasoactive intestinal polypeptide stimulation of protein secretion from rat lacrimal gland acini, Am. J. Physiol., 247 (1984) G502-G509. [10] Dartt, D.A., Shulman, M., Gray, K.L., Rossi, S.R., Matkin, C. and Gilbard, J.P., Stimulation of rabbit lacrimal gland secretion with biologically active peptides, Am. J. Physiol., 254 (1988) G300G306. [11] Ennerback, L., Mast cell heterogeneity: the evolution of the concept of a specific mucosal mast cell. In A.D. Befus, J. Bienenstock and J.A. Denburg (Eds.), Mast cell differentiation and heterogeneity. Raven Press, New York, 1986, pp. 1-26. [12] Foreman, J.C., Peptides and neurogenic inflammation, Brit. Med. Bull., 43 (1987) 386-392. [13] Galli, S.J., New insights into "the riddle of the mast cells': microenvironmental regulation of mast cell development and phenotypic heterogeneity, Lab. Invest., 62 (1990) 5-33. [14] Herzog, V. and Fahimi, H., A new sensitive colorimetric assay lor peroxidase using 3,3'-diaminobenzidine as a hydrogen donor, Analyt. Biochem., 55 (1973) 554-562. [15] Holzer, P., Local effector functions of capsaicin-sensitive sensory nerve endings: Involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides, Neuroscience, 24 (1988) 739-768. [16] Hussain M. and Singh, J., Is VIP the putative non-cholinergic, non-adrenergic neurotransmitter controlling protein secretion in rat lacrimal glands? Quart. J. Exp. Physiol., 73 (1988) 135-138. [17] Jones, L.T., The lacrimal secretory system and its treatment, Am. J. Ophthalmol., 62 (1966) 47-60. [18] Kelleher, R.S., Hann, L.E., Edwards, J.A. and Sullivan. D.A., Endocrine, neural, and immune control of secretory component output by lacrimal gland acinar cells, J. Immunol., 146 (1991) 3405 -3412. [19] Kiernan, J.A., The effects of known and suspected neurotransmitter substances and of some nucleotides on isolated mast cells, Experientia, 28 (1972) 653-655. [20] Krumins, S.A. and Broomfield, C.A.. Evidence of NK1 and NK2 tachykinin receptors and their involvement in histamine release in a murine mast cell line, Neuropeptides, 21 (1992) 65-72.
[21] Krumins, S.A. and Broomfield, C.A., C-terminal substance P fragments elicit histamine release from a murine mast cell line, Neuropeptides, 24 (1993) 5-10. [22] Matsumoto, Y., Tanabe, T., Ueda, S. and Kawata, M., lmmunohistochemical and enzymehistochemical studies of peptidergic, aminergic and cholinergic innervation of the lacrimal gland of the monkey (Macaca fuscata), J. Autonom. Nerv. Syst., 37 (1991) 207-214. [23] Nikkinen, A,, Lehtosalo, J.l., Uuistalo, H., Palkama, A. and Panula, P., The lacrimal glands of the rat and the guinea pig are innervated by nerve fibers containing immunoreactivities for substance P and vasoactive intestinal polypeptide, Histochemistry, 81 (1984) 23-27. [24] Nikkinen, A., Uuitalo, H., Lehtosalo, J. and Palkama, A., Distribution of adrenergic nerves in the lacrimal glands of guinea pig and rat, Exp. Eye Res., 40 (1985) 751-756. [25] Pariente, J.A., Madrid, J.A. and Salido, G.M., Histamine receptors in unstimulated pancreatic exocrine secretion of the rabbit, Agents Actions, 28 (1989) 62-69. [26] Piotrowski, W. and Foreman, J.C., On the actions of substance P, somatostatin and vasoactive intestinal polypeptide on rat peritoneal mast cells and in human skin, Arch. Pharmacol., 331 (1985) 364368. [27] Repke, H. and Bienert, M., Mast cell activation - - a receptor-independent mode of action of substance P? FEBS Lett., 221 (1987) 236-240. [28] Rossi, S.R, Dartt, D.A. and Gilbard, J.P., Eledoisin and lacrimal secretion in the rabbit, Curr. Eye Res., 9 (1990) 273-276. [29] Salido, G., Lennard, R., Singh, J. and Pariente, J.A., Histamineevoked amylase secretion is associated with small changes in calcium mobilization in isolated guinea-pig pancreas, Exp. Physiol., 75 (1990) 263-266. [30] Shanahan, F. and Anton, P., Neuroendocrine modulation of the immune system. Possible implications for inflammatory bowel disease, Dig. Dis. Sci., 33 (3 Suppl.) (1988) 41S-49S. [31] Sharkey, K.A., Substance P and calcitonin gene-related peptide (CGRP) in gastrointestinal inflammation, Ann. N.Y. Acad. Sci., 664 (1992) 425-442. [32] Sharkey, K.A. and Pittman, Q.J., The autonomic nervous system: peripheral and central integrative aspects. In R. Greger and U. Windhorst (Eds.), Human physiology - - from cellular mechanisms to integration, Springer Verlag, Heidelberg and New York, in press. [33] Sibony, P.A., Walcott, B., Mckeon, C. and Jakobiec, F.A., Vasoactive intestinal polypeptide and the innervation of the human lacrimal gland, Arch. Ophthamol., 106 (1988) 1085-1088. [34] Stead, R.H., Perdue, M.H., Blennerhassett, M.G., Kakuta, Y., Sestini, P. and Bienenstock, J., The innervation of mast cells. In S. Freier (Ed.), Neuroendocrine-immune network, CRC Press, Boca Raton, 1990, pp. 19-37. [35] Uddman, R., Alumets, J., Ehinger, B., Hakanson, R., Loren, I. and Sundler, F., Vasoactive intestinal peptide nerves in ocular and orbital structures of the cat, Invest. Ophthalmol. Vis. Sci., 19 (1980) 878-885. [36] Weihe, E., Muller, S., Fink, T. and Zentel, H.J., Tachykinins, calcitonin gene-related peptide and neuropeptide Y in nerves of the mammalian thymus: interactions with mast cells in autonomic and sensory neuroimmunomodulation? Neurosci. Lett., 100 (1989) 7782. [37] White, M.V., Mast cell secretagogues. Histamine releasing factors and neuropeptides. In M.A. Kaliner and D.D. Metcalfe (Eds.), The mast cell in health and disease. Marcel Dekker Inc., New York, 1993, pp. 109-128. [38] Williams, R.M., Sharkey, K.A., Lea, R.W. and Singh, J., Interaction between peptides and mast cells in the control of peroxidase secretion from the rat isolated lacrimal gland, J. Physiol. (Lond.), 477 (1994) 66P. [39] Williams, R.M., Singh, J. and Sharkey, K.A., Innervation and mast cells of the rat exorbital lacrimal gland: the effects of age, J. Auton. Nerv. Syst., 47 (1993) 95-108.
ELSEVIER
Journal of the Autonomic Nervous System 61 (1996) 37-42
Autonomic Nervous System
Secretion and serotonin release in the isolated rat lacrimal gland: the effects of substance P and calcitonin gene-related peptide Ruth M. Williams a.b, Lorenzo Bauce a, Robert W. Lea h, Jaipaul Singh b, Keith A. Sharkey a,* a Neuroscience Research Group, Department of Medical Physiology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada b Cell Communication Group, Department of Applied Biology, University of Central Lancashire, Preston PR1 2HE, UK Received 1 September 1995; revised 17 January 1996; accepted 27 January 1996
Abstract A close anatomical relationship between nerves containing substance P and caicitonin gene-related peptide (CGRP) and mast cells containing serotonin has been demonstrated in the rat lacrimal gland. This study investigates the potential for peptidergic regulation of lacrimal mast cells by examining the actions of substance P, CGRP and serotonin on protein and peroxidase secretion from isolated lacrimal segments and on substance P and CGRP to release serotonin from the lacrimal mast cells. Substance P, CGRP and serotonin evoked marked increases in total protein and peroxidase from the lacrimal. Sodium cromoglycate, a mast cell stabilizer, significantly reduced or blocked the secretory responses elicited by these agonists. Chromatographic analysis using electrochemical detection revealed Ihat substance P, but not CGRP, augmented the release of serotonin from the gland. The substance P evoked peroxidase secretion and serotonin release was blocked by CGRP and by sodium cromoglycate. These results support a role for mast cells in the regulation of lacrimal secretion and suggest a novel regulatory interaction between substance P and CGRP in the control of lacrimal function through a meuro-immune interaction. Keywords: Sodium cromoglycate; Mast cells; Substance P; 5-HT; CGRP; Neuroimmunology
1. Introduction The secretion of tears by the exorbital lacrimal gland is primarily under the control of the autonomic nervous system [2,6,17]. Secretion elicited by electrical field stimulation of isolated lacrimal glands was shown to be in part resistant to combined autonomic blockade [16], suggesting a non-adrenergic, non-cholinergic (NANC) innervation of the gland. These NANC nerves were believed to be peptidergic and immunohistochemical studies have since revealed an extensive peptidergic innervation of the lacrimal in several species, including man [9,22-24,33,35,39]. The precise physiological role of many of these peptides has yet to be defined, but it is suggested that some may function as secretomotor transmitters in the regulation of lacrimation [9,10,18].
* Corresponding author. Tel.: + 1 403 2204601; fax: + 1 403 2838731; e-mail: ksharkey @acs.ucalgary.ca
In a recent study of the rat lacrimal it was demonstrated that nerves containing substance P and calcitonin gene-related peptide (CGRP) were closely associated with mast cells in the gland [39]. Elsewhere, such as the skin and GI tract, a close association between mast cells and peptidergic nerves has been reported [34,36]. These peptides have previously been studied for their potential role in the modulation of mast cell secretion and more generally in the modulation of immune cell activity [30,31,34,37]. Mast cells have a secretory capacity. In mouse and rat, the released mediators include the biogenic amines histamine and serotonin (5-HT), which have pronounced biological effects that include exocrine secretion from glands [18,8,13,25,29,37]. Classically, the release of mast cell mediators is induced by an antigen-antibody (IgE) reaction on the mast cell membrane [1,13]. Recent evidence suggests that neural mechanisms are also involved in mast cell stimulation. It is now apparent that neuropeptides can modulate some immune responses directly (see [37] for a review) and several peptides, notably substance P, can modulate mast cell mediator release [1,12,13,37]. Since
0165-1838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII SO 165-1 838(96)0003 1-8
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R.M. Williams et al. / Journal of the Autonomic' Nervous System 61 (1996) 37-42
mast cell mediators and neurotransmitters can potentially elicit lacrimal secretion and since there is evidence for a functional association between nerves and mast cells in the skin [12,26,34], we sought to test the hypothesis that there is a functional relationship between nerves and mast cells in the rat lacrimal gland. To do this we examined the action of substance P and CGRP in eliciting secretion in the isolated lacrimal gland in the presence and absence of the mast cell membrane stabilizer sodium cromoglycate. Since we had evidence that 5-HT was present in mast cells in the rat lacrimal [39] we also studied the effects of 5-HT on secretion. Finally, we measured the effects of the substance P and CGRP on 5-HT release from isolated glands. A preliminary account of part of this work has been previously published [38].
2. Materials and methods 2.1. Animals
All the procedures described below were approved by institutional (Canada) and national (UK) Animal Care Committees. Experiments were performed on adult male Sprague-Dawley rats (200-250 g; Charles River, Quebec, Canada; Charles River, Sussex, UK). Rats were housed under constant photoperiod (14 h light, 10 h dark) and temperature (20°C) and were allowed food and water ad libitum. 2.2. Tissue preparation
Rats were killed by cervical dislocation and exsanguination. Lacrimal glands were quickly excised and placed in a modified oxygenated (95% 0 e / 5 % CO 2) Krebs-Henseleit (KH) solution of the following composition (mM): NaCI, 118; KC1, 3.7; CaC12, 2.56; MgCI 2, 1.2; NaHCO 3, 25; KH2PO 4, 2.2; D-glucose, 10; pH 7.3-7.4. The connective tissue capsule was carefully and completely removed from each gland, which was then sliced into small segments (5-10 mg). In each experiment about 25% of a gland was used (30-35 mg). Lacrimal segments were washed (5 times) with KH solution and placed in 5 ml plastic tubes containing either KH solution alone (control) or KH solution containing different concentrations of test substances. Segments were incubated for a basal period of 20 min at 37°C in a shaking water bath, then KH was removed and replaced with the same volume of KH containing the test compound. Tissues were incubated for a further 20 min. At the end of the final incubation period, samples were collected and analyzed as described below. In studies using sodium cromoglycate (a gift from Fisons Pharmaceuticals, UK), tissues were preincubated for an additional 15 min prior to the addition of agonists which were incubated for 20 min as previously described. In each case, tissues were tested with only a single concentration of agonist in the
presence or absence of sodium cromoglycate. At the end of each experiment tissues were removed, blotted dry and weighed. 2.3. Peroxidase assay
Peroxidase activity was measured as a marker of acinar cell secretion using a modification of the method of Herzog and Fahimi [14]. Briefly, 200 I~1 of the incubation medium was added to 3,3'- diaminobenzidine (5 raM) in sodium phosphate buffer (0.1 M, pH 7.4). Hydrogen peroxide was added and the contents mixed. The optical density was measured immediately at 460 nm using a Visi spectrophotometer (Pharmacia, UK). Absorbance was measured over 3 min and the slope of the measurements used to determine peroxidase activity using a peroxidase (Sigma) standard and KH solution as a blank. All values are expressed as ng ml-1 100 rag-J tissue. 2.4. Protein assay
The protein content of the samples was determined spectrophotometrically using a modification of the Bradford assay [3], described by Bromberg and Welch [7]. Samples of incubation medium were added to the protein reagent and mixed. Optical density was recorded at 595 nm using a Visi spectrophotometer (Pharmacia, UK). Absorbance was measured after 30 min using KH solution as a blank and bovine serum albumin (Sigma) as a standard. All assays were performed in duplicate. Concentrations were determined from standard curves constructed for each series of experiments. Protein secretion was expressed as m g m l J 100mg ~ tissue. 2.5. Serotonin release
Perchloric acid (100 pA) was added to a sample of the incubation medium (900 Ixl) to precipitate protein. The mixture was shaken and left for 10 min on ice. After centrifugation (10,000 × g for 1 min), the supernatant was analyzed for 5-HT by high performance liquid chromatography (HPLC) using an electrochemical detection system. Known concentrations of 5-HT (Sigma) were used as standards. Samples were separated on a Beckman Ultrasphere Octadecylsilica column (4.5 × 50 mm, 5 Ixm) using a mobile phase consisting of 0.2 M acetic acid, 0.05 M sodium acetate, 100 m g / l EDTA, 100 mg/1 sodium dodecylsulphate and 25% methanol ( v / v ) at a flow rate of 0.5 m l / m i n at 40°C. Detection was by means of a BAS LC4B amperometric detector with a glassy carbon electrode operating at +0.60 V. The signal was digitized, stored and quantitated using the external standard method using a Waters Maxima data acquisition system. Results are expressed as pmol 100 mg -~ tissue. In order to assess the total 5-HT content of the lacrimal, glands were homoge-
39
R.M. Williamset al. / Journal of the Autonomic Nervous System 61 (1996) 37-42
a i z e d in perchloric acid and the supernatant analyzed as described above.
A. A 0
Ji"
2.6. Statistical analysis
!-
Data are presented as mean ___ standard error o f the mean (SEM) from 5 to 8 experiments using glands from different animals. Data were compared using A N O V A followed by N e w m a n - K e u l s post hoc test, or unpaired Student's t-tests as appropriate. Differences between values were considered significant at P < 0.05.
3.1. Lacrimal secretion
Two measures o f lacrimal secretion were used in the Wesent study: peroxidase release and total protein release. In all cases, parallel effects were observed with either assay, where comparable experiments were performed. Basal secretion of protein (measured in one series of ~ltperiments) was 1.46-t-0.1 mg ml - I 100 mg -= tissue ( n = 6) and basal peroxidase secretion 32.8 + 4.4 ng m l - 1 190 mg - ] tissue (n = 24).
2-
c
"
-, ......... 10-9
, ........ . ........ . 10-8 1 0 -), 10-6
Substance P a.
3. Results
4-
. . . . . . . .
i
.
.
10-~J
(M)
A
E; '6,:'.
substance P
substance ÷ Na
P
cromoglycate
Fig. 1. (A) The effect of varying concentrations of substance P on protein release from isolated segments of lacrimal gland. Substance P caused a concentration-dependent increase in protein release (basal subtracted) at concentrations above 10 -9 M, with a maximal effect at about 10 - 6 M. (B) The effect of sodium cromoglycate (10 -4 M) on substance P (10 - 6 M) induced protein release. Sodium cromoglycate virtually abolished protein release (*, P < 0.01) evoked by substance P. Each point is a mean of 5-8 responses from different glands.
3,2. Substance P Substance P evoked a concentration-dependent protein release with a maximal effect at 10 -6 M and an ECs0 of 2 X 10 -8 M (Fig. 1). Similarly, peroxidase release was enhanced over a similar concentration range and EC50 (7 × 10 -8 M)(Fig. 2). In both cases the response to substance P was blocked by prior addition of the mast cell stabilizer sodium cromoglycate (10 -4 M). This concentration of sodium cromoglycate had no significant effect on basal secretion of either protein or peroxidase (data not shown).
3.4• Serotonin
Since 5-HT has been reported to be a secretagogue in other exocrine glands and is stored in mast cells of the lacrimal gland we examined the effects of 5-HT on protein and peroxidase secretion. 5 - H T caused a concentrationdependent peroxidase release at a somewhat reduced potency compared to either substance P or C G R P (EC50 5 X 10 -7 M) (Fig. 4). Protein secretion was also enhanced
100-
3,3. CGRP
C G R P evoked protein release (at the concentration tested (10 -7 M)), and at this and higher concentrations a peroxidase secretion, similar in magnitude to that evoked by equimolar concentrations o f substance P (Fig. 3). The BC50 of the response (9 x 10 -8 M) was not significantly different from that of substance P. The effects of combinillg substance P and C G R P were examined for peroxidase secretion. It was found that at the two concentrations tested, there was a marked attenuation in the effect of C G R P in the presence of substance P (Fig. 3). The effect o f sodium cromoglycate was examined only for protein secretion. In this case, there was no effect o f the mast cell stabilizer ( P = 0.17) on the C G R P evoked protein secretion.
J!
o Krebs
50-
•
Substance Substance
P P +
Na Cromoglycate ' ,"o-; "~o-; "i'o-; "~'o-' 'i~-; 'i~-' Substance P [M]
Fig. 2. The effect of varying concentrations of substance P (filled squares) on peroxidase release from isolated segments of lacrimal gland. Substance P caused a concentration-dependent increase in peroxidase release above basal (basal shown by the open circle) at concentrations above 10-s M, with a maximal effect at about 5 X 10 -6 M. The effect of sodium cromoglycate (10 -4 M) on substance P-induced peroxidase release (filled triangles). Sodium cromoglycate abolished peroxidase release (*, P < 0.05, ANOVA) evoked by substance P. Each point is a mean of 5-8 responses from different glands.
R.M. Williams et al. / Journal c)[the Autonomic Nervous System 61 (1996) 37-42
40 A.
100-
75"
50"
• CGRP
o ~ E-
~g
by 5-HT at the concentration tested (10 _5 M, which caused maximal peroxidase release). This effect was substantially reduced by pretreatment of the tissue by sodium cromoglycate, suggesting that 5-HT was acting to degranulate mast cells to cause its effect.
• CGRP + substance p
25"
3.5. Serotonin release . . . . . . . . . . . . . . . .
' 1~.~ o ' "
0
lO.O
I
. . . . . . .
lO.S
I
. . . . . . . . . . .
10.7
10.6
CGRP (M)
B.
CGRP
CGRP
+ Na cromoglycate
Fig. 3. (A) The effect of varying concentrations of CGRP (filled squares) on peroxidase release from isolated segments of lacrimal gland. CGRP caused a concentration-dependent increase in peroxidase release at concentrations above 10 -8 M. Equimolar mixtures of CGRP and substance P (filled triangles) blocked the peroxidase release (*, P < 0.05). (B) The effect of sodium cromoglycate (10 -4 M) on CGRP (10 7 M)-induced protein release (basal subtracted). Sodium cromoglycate had no effect on protein release evoked by CGRP. Each point is a mean of 5 - 8 responses from different glands.
A.
Mean basal release of 5-HT was 4.3 _+ 0.4 pmol 100 mg -t tissue (n = 15). Chromatographic analysis of the effluent samples in response to substance P demonstrated that substance P evoked a two-fold increase in 5 - HT release at concentrations above 10 8 M (10 8 M, 9.9 + 0.7 pmol 100mg ~ tissue; 10 6 M, 9 . 2 _ + 0 . 5 p m o l l 0 0 m g - t tissue). Lower concentrations (10 1 0 - 1 0 - 9 M ) failed to stimulate 5-HT release. Pretreatment of the tissue with sodium cromoglycate (10 4 M) significantly reduced, to slightly below basal levels, 5-HT release in response to 10 - 6 M substance P (3.0 + 0.1 pmol 100 mg -~ tissue). CGRP was without effect on 5-HT release at concentrations of 10 1 0 - 1 0 - 6 M and indeed at the highest concentration (10 6 M) levels were significantly below basal (2.5 _+ 0.4 pmol 100 m g - ] tissue, P < 0.05). As for peroxidase secretion, addition of CGRP blocked the effects of substance P ( 1 0 - 6 M substance P + 10 6 M CGRP; 3.45 _+ 0.4 pmol 100 mg l). The total content of 5-HT in the rat lacrimal gland was 9 5 + 4 . 5 pmol 100 mg a tissue ( n = 5 ) . Based on this data it appears that substance P maximally stimulated the release of about 9% of the total glandular 5-HT content.
15o.
4. Discussion x ~. o-~
o
io-;*'
1o-'
1o"
to-'
1o"
lo-'
1o-'
5-HT (M) B.
|
5-HT
5-HT
Na c r o ~ g l y c a t e
Fig. 4. (A) The effect of varying concentrations of 5-HT (filled squares) on peroxidase release from isolated segments of lacrimal gland. 5-HT caused a concentration-dependent increase in peroxidase release at concentrations above 10 - s M, with a maximal effect at about 10 5 M. (B) The effect of sodium cromoglycate (10 -4 M) on 5 - H T (10 5 M)-induced protein release (basal subtracted). Sodium cromoglycate significantly reduced ( *, P < 0.05) protein release evoked by 5-HT. Each point is a mean of 5 - 8 responses from different glands.
Exogenous application of substance P to isolated lacrimal segments resulted in a concentration-dependent peroxidase and protein secretion. This secretory response could be almost totally abolished by pretreatment of the tissue with the mast cell stabilizer sodium cromoglycate. Application of CGRP and 5-HT to isolated lacrimal segments evoked a concentration-dependent peroxidase release and significant protein secretion. Whereas secretion elicited by 5-HT could be attenuated by sodium cromoglycate, that evoked by CGRP was not blocked. These findings implicate the mast cell as an intermediary in the regulation of lacrimation by neuropeptides. Furthermore they suggest a role for mast cell mediators in tear secretion and their potential modulation by nerves containing substance P and CGRP. These findings extend the anatomical observations previously presented [39], demonstrating a close relationship between 5-HT-containing mast cells and nerve fibers containing substance P and CGRP in the rat lacrimal gland. Substance P has previously been demonstrated to degranulate isolated mast cells obtained from different sources
R.M. Williams et al. / Journal of the Autonomic Nervous System 61 (1996) 37-42
representing the two heterogeneous classes of mast cell described in rats (connective tissue [peritoneal] type mast cells and mucosal mast cells) [11,13,37]. In addition, studies in vivo show that substance P can degranulate mast cells in the skin [26]. The mechanism of action of substance P has previously been described as a receptor-independent pathway [27], which may in part explain the lack of concentration-responsiveness of 5-HT release. More te.cently, however, this idea has been challenged suggesting that substance P can act via NK1 and NK2 neurokinin receptors on mast cells [20,21]. CGRP is considerably less effective as a mast cell ~,cretagogue when compared to substance P [12,31]. However, previous studies have shown that substance P can attenuate the action of CGRP through the release of mast Gell proteases [4,5]. In this study not only was CGRP unable to stimulate 5-HT release, its agonist action on protein release was unaffected by a mast cell stabilizer. Xowever, CGRP was apparently able to interact with substance P to effectively prevent the action of substance P. The mechanism whereby CGRP exerts its inhibitory actions is not apparent from this study, but these results ~uggest a possible new regulatory interaction between these two peptides in lacrimal physiology. In the lacrimal gland the role of 5-HT has not previously been examined. Based on immunohistochemical ob~rvations the sole source of 5-HT in the gland is mast qells [39]. Consistent with this observation, the mast cell ~labilizer sodium cromoglycate effectively suppressed the 5-HT release induced by substance P and reduced protein release induced by substance P and 5-HT, but not CGRP. This suggests that the protein secretory effect of substance P and 5-HT is indirectly mediated by mast cells, whose released mediators give rise to lacrimal secretion. Why the ~ f e c t of substance P on 5-HT release is not ooncentration-dependent is unclear. As mentioned above, it may degranulate mast cells in a receptor-independent manaer [27]. Also, the apparent lack of concentration-responsiveness might be explained by rapid uptake and degradation mechanisms for 5-HT that exist and that mast cell degranulation may not be an entirely graded phenlomenon. The concentration-dependency of the secretion data may then be due to actions of the peptide on mast cells and the net effect of the mast cell mediators being a ~lraded response through activation of receptors on the acinar cells of the lacrimal. Which mast cell mediator gives rise to secretion in tmknown, but histamine and 5-HT are recognized in other e~xocrine glands as secretagogues [8,25,29]. The capacity of 5-HT to degranulate mast cells has previously been examreed [19]. Serotonin was found not to degranulate rat ~ritoneal mast cells in vitro; however, the staining characteristics of lacrimal mast cells are not entirely consistent with them being of this type [39]; hence interpretation of the effects of 5-HT on lacrimal mast cells requires further investigation.
41
The mast cell-nerve relationship suggested by the results of the present study is worthy of additional study, since several important questions remain unanswered. Firstly, is this an obligatory functional unit, such that the release of substance P always causes mast cell activation? If that is the case, and substance P is in primary afferent nerves, then is there a neurogenic lacrimation analogous to neurogenic inflammation in the skin [15]? In this context, sensory reflex lacrimation stimulated by a substance P analogue (eledoisin) has been reported in the rabbit [28], but the same compound applied intraarterially to the lacrimal failed to elicit lacrimal secretion [10]. Conversely, is there bidirectional communication and can mast cells activate nerves in the gland? In either case answers to these questions are important in the context of allergy, as tear production may be affected in situations where mast cell degranulation occurs, e.g. in certain allergies. Previous studies in the lacrimal gland have suggested an endocrine, neural and immune control of the release of secretory component from isolated acinar cells [18]. Secretory component is the lgA receptor and functions to transfer IgA into tears. In this study substance P and CGRP were found not to stimulate (or inhibit) secretory component, whereas vasoactive intestinal polypeptide stimulated its release and a cholinergic agonist inhibited its release [18]. Certain cytokines also stimulated the release of secretory component (interleukin 1 and tumor necrosis factor). Since cytokines are released from mast cells [1,13], it is possible that under conditions where mast cells are stimulated (such as the present study) secretory component is also released in association with other proteins to effect the protective actions that they serve in the normal physiology of the eye. In conclusion, the present study demonstrates that neuro-immune interactions are probably a normal part of lacrimal secretory physiology or pathophysiology, as is being increasingly recognized as a fundamental feature of most autonomically innervated tissues [32,36]. The added complexity endows the autonomic nervous system with additional roles that suggests it functions in a more integrative manner than was previously suspected. Finally, a better understanding of the interactions between the nervous and immune systems in the lacrimal gland may offer novel avenues for therapeutic intervention in diseases such as keratoconjunctivitis sicca and other diseases of aqueous tear film deficiency.
Acknowledgements This work was supported by the Medical Research Council of Canada (grant to KAS), The British Council Academic Links Scheme (to K.A.S. and J.S.), and the Wellcome Trust (grant to J.S.). K.A. Sharkey is an Alberta Heritage Foundation for Medical Research Scholar. R.M. Williams received a Studentship from the Science and
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R.M. Williams et al. / Journal of the Autonomic Nervous System 61 (1996) 37-42
Engineering Research Council. We thank Fisons Pharmaceuticals (UK) for generously providing the sodium crom o g l y c a t e u s e d in this s t u d y .
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[21] Krumins, S.A. and Broomfield, C.A., C-terminal substance P fragments elicit histamine release from a murine mast cell line, Neuropeptides, 24 (1993) 5-10. [22] Matsumoto, Y., Tanabe, T., Ueda, S. and Kawata, M., lmmunohistochemical and enzymehistochemical studies of peptidergic, aminergic and cholinergic innervation of the lacrimal gland of the monkey (Macaca fuscata), J. Autonom. Nerv. Syst., 37 (1991) 207-214. [23] Nikkinen, A,, Lehtosalo, J.l., Uuistalo, H., Palkama, A. and Panula, P., The lacrimal glands of the rat and the guinea pig are innervated by nerve fibers containing immunoreactivities for substance P and vasoactive intestinal polypeptide, Histochemistry, 81 (1984) 23-27. [24] Nikkinen, A., Uuitalo, H., Lehtosalo, J. and Palkama, A., Distribution of adrenergic nerves in the lacrimal glands of guinea pig and rat, Exp. Eye Res., 40 (1985) 751-756. [25] Pariente, J.A., Madrid, J.A. and Salido, G.M., Histamine receptors in unstimulated pancreatic exocrine secretion of the rabbit, Agents Actions, 28 (1989) 62-69. [26] Piotrowski, W. and Foreman, J.C., On the actions of substance P, somatostatin and vasoactive intestinal polypeptide on rat peritoneal mast cells and in human skin, Arch. Pharmacol., 331 (1985) 364368. [27] Repke, H. and Bienert, M., Mast cell activation - - a receptor-independent mode of action of substance P? FEBS Lett., 221 (1987) 236-240. [28] Rossi, S.R, Dartt, D.A. and Gilbard, J.P., Eledoisin and lacrimal secretion in the rabbit, Curr. Eye Res., 9 (1990) 273-276. [29] Salido, G., Lennard, R., Singh, J. and Pariente, J.A., Histamineevoked amylase secretion is associated with small changes in calcium mobilization in isolated guinea-pig pancreas, Exp. Physiol., 75 (1990) 263-266. [30] Shanahan, F. and Anton, P., Neuroendocrine modulation of the immune system. Possible implications for inflammatory bowel disease, Dig. Dis. Sci., 33 (3 Suppl.) (1988) 41S-49S. [31] Sharkey, K.A., Substance P and calcitonin gene-related peptide (CGRP) in gastrointestinal inflammation, Ann. N.Y. Acad. Sci., 664 (1992) 425-442. [32] Sharkey, K.A. and Pittman, Q.J., The autonomic nervous system: peripheral and central integrative aspects. In R. Greger and U. Windhorst (Eds.), Human physiology - - from cellular mechanisms to integration, Springer Verlag, Heidelberg and New York, in press. [33] Sibony, P.A., Walcott, B., Mckeon, C. and Jakobiec, F.A., Vasoactive intestinal polypeptide and the innervation of the human lacrimal gland, Arch. Ophthamol., 106 (1988) 1085-1088. [34] Stead, R.H., Perdue, M.H., Blennerhassett, M.G., Kakuta, Y., Sestini, P. and Bienenstock, J., The innervation of mast cells. In S. Freier (Ed.), Neuroendocrine-immune network, CRC Press, Boca Raton, 1990, pp. 19-37. [35] Uddman, R., Alumets, J., Ehinger, B., Hakanson, R., Loren, I. and Sundler, F., Vasoactive intestinal peptide nerves in ocular and orbital structures of the cat, Invest. Ophthalmol. Vis. Sci., 19 (1980) 878-885. [36] Weihe, E., Muller, S., Fink, T. and Zentel, H.J., Tachykinins, calcitonin gene-related peptide and neuropeptide Y in nerves of the mammalian thymus: interactions with mast cells in autonomic and sensory neuroimmunomodulation? Neurosci. Lett., 100 (1989) 7782. [37] White, M.V., Mast cell secretagogues. Histamine releasing factors and neuropeptides. In M.A. Kaliner and D.D. Metcalfe (Eds.), The mast cell in health and disease. Marcel Dekker Inc., New York, 1993, pp. 109-128. [38] Williams, R.M., Sharkey, K.A., Lea, R.W. and Singh, J., Interaction between peptides and mast cells in the control of peroxidase secretion from the rat isolated lacrimal gland, J. Physiol. (Lond.), 477 (1994) 66P. [39] Williams, R.M., Singh, J. and Sharkey, K.A., Innervation and mast cells of the rat exorbital lacrimal gland: the effects of age, J. Auton. Nerv. Syst., 47 (1993) 95-108.