Naunyn-Schmiedeberg's Arch Pharmacol (2004) 370: 305–313DOI 10.1007/s00210-004-0972-z

ORIGINAL ARTICLE

Michel Bernhard . Kenneth Takeda . Caroline Keller .Mirko Haslebacher . George N. Lambrou .Anne-Ulrike Trendelenburg

3H-noradrenaline release from mouse iris–ciliary body:role of presynaptic muscarinic heteroreceptors

Received: 20 April 2004 / Accepted: 27 July 2004 / Published online: 16 September 2004# Springer-Verlag 2004

Abstract Sympathetic neurotransmitter release and itsmodulation by presynaptic muscarinic heteroreceptorswere studied in mouse iris–ciliary bodies. Tissue prepara-tions were preincubated with 3H-noradrenaline and thensuperfused and stimulated electrically. Firstly, experimen-tal conditions were defined, allowing study of presynapticsympathetic inhibition in mouse iris–ciliary body. If tissuewas stimulated four times with 36 pulses/3 Hz, tritiumoverflow peaks were reliably and reproducibly measured.As expected, these stimulation conditions led to markedα2-autoinhibition as indicated by the release-enhancingeffect of the α2-antagonists phentolamine and rauwols-cine. To ensure autoinhibition-free 3H-noradrenalinerelease, which is optimal for studying presynaptic sympa-thetic inhibition, α2-receptors were blocked in allsubsequent experiments. Under these conditions, evokedtritium overflow was almost completely abolished in thepresence of the sodium channel blocker tetrodotoxin,indicating a neuronal origin of 3H-noradrenaline release.Secondly, muscarinic inhibition of 3H-noradrenalinerelease was characterized using the conditions describedabove (36 pulses/3 Hz; phentolamine 1 μM andrauwolscine 1 μM throughout). The muscarinic receptoragonist oxotremorine M decreased evoked tritium over-flow in a concentration-dependent manner with an IC50 of0.33 μM and maximal inhibition of 51%. The concentra-tion–response curve of oxotremorine M was shifted to theright by the muscarinic antagonists ipratropium andmethoctramine, whereas pirenzepine was ineffective. Theobserved rank order of antagonist potencies, ipratropium >

methoctramine > pirenzepine, which is typical for the M2

subtype, indicates that presynaptic muscarinic receptors onsympathetic axons of mouse iris–ciliary bodies arepredominantly M2. Finally, inhibition of 3H-noradrenalinerelease by endogenously secreted acetylcholine wasinvestigated. Longer pulse trains, 120 pulses/3 Hz and600 pulses/5 Hz, were used and the cholinesteraseinhibitor physostigmine was added to the superfusionmedium to increase synaptic levels of endogenous acetyl-choline. Under these conditions, ipratropium approxi-mately doubled the evoked overflow of tritium, indicatingthat endogenously released acetylcholine can activatepresynaptic muscarinic heteroreceptors. In conclusion,the present experiments establish measurement of theelectrically induced release of 3H-noradrenaline frommouse iris–ciliary bodies. As in other species, noradren-aline release in this preparation was subject to presynapticmuscarinic inhibition. Our results also indicate that thepresynaptic muscarinic receptors on sympathetic axons inmouse iris–ciliary body are predominantly M2. Moreover,these receptors can be activated by both exogenousagonists and endogenously released acetylcholine and,hence, may operate physiologically in the interplaybetween the parasympathetic and sympathetic nervoussystem.

Keywords Mouse iris–ciliary body . 3H-noradrenalinerelease . Presynaptic muscarinic receptors . Muscarinicreceptor subtypes . Sympathetic axon terminals

Introduction

Various ocular functions are controlled by the sympatheticnervous system. Amongst these are, for example, contrac-tion of iris smooth muscles which controls pupil diameterand production of aqueous humor in ciliary body epithe-lium which regulates intraocular pressure. One possibilityto influence sympathetically controlled functions, activelyused in pharmacotherapy, is by activation or blockade ofpresynaptic receptors which are responsible for the fine-

M. Bernhard . C. Keller . M. Haslebacher . G. N. Lambrou .A.-U. Trendelenburg (*)DA Ophthalmology, Novartis Institutes for BioMedicalResearch,PO Box WSJ-386.746, 4002 Basel, Switzerlande-mail: anne-ulrike.trendelenburg@pharma.novartis.comTel.: +41-61-3243710Fax: +41-61-3246458

M. Bernhard . K. TakedaCNRS UMR7034, Université Louis Pasteur,67404 Illkirch, France

tuning of sympathetic neurotransmission (Langer 1997;Boehm and Kubista 2002). Some antiglaucomatous drugs(e.g., the α2-adrenoceptor agonist brimonidine) arebelieved to act at least partly via presynaptic sympatheticinhibition in the iris–ciliary body (Burke and Potter 1986).Moreover, miosis observed after administration of pilo-carpine is also thought to be the result of presynapticsympathetic inhibition (Bognar et al. 1988; Nietgen et al.1999). More precise knowledge concerning the occurrenceand nature of presynaptic receptors on sympathetic axonsin the iris–ciliary body may, thus, serve as a basis todiscover innovative and more specific targets for pharma-cological interventions.

Sympathetic transmitter release and its modulation bypresynaptic receptors have been studied for more than30 years in iris–ciliary body preparations from severalspecies including man (Farnebo and Hamberger 1970;reviewed by Fuder 1994). The existence of release-inhibiting α2-autoreceptors has been well demonstrated(Farnebo and Hamberger 1970, 1971; Jumblatt et al. 1987;Jumblatt 1994; Opere and Ohia 1998). Moreover, variousrelease-inhibiting heteroreceptors have been described inthis tissue, including adenosine A1 (Fuder et al. 1992;Crosson and Gray 1997), purinergic P2Y-like (Fuder et al.1992; Fuder and Muth 1993), neuropeptide Y (Ohia andJumblatt 1990b), dopamine D2-like (Ogidigben et al.1993; Chu et al. 1999), muscarinic M2-like (Jumblatt andNorth 1988; Bognar et al. 1988, 1989, 1992; Fuder et al.1989; Dammann et al. 1989; Jumblatt and Hackmiller1994), histamine H3 (Koss and Hey 1993; Kulkarni et al.2002), prostaglandin (Neufeld and Page 1975; Ohia andJumblatt 1990a, 1991), cannabinoid CB1 (Shara et al.2002), serotonin 5-HT1 (Harris et al. 2002) and opioidreceptors (Drago et al. 1980; Russel and Potter 2001). Inaddition, release-facilitating receptors were described iniris–ciliary body preparations, including angiotensin II(Jumblatt and Hackmiller 1990; Ohia and Jumblatt 1993),adenosine A2 (Fuder et al. 1992) and serotonin 5-HT3

receptors (Harris et al. 2002; see also Jumblatt et al. 1993;Fuder 1994; Boehm and Kubista 2002).

While only limited information is available about thesubtypes of presynaptic receptors on noradrenergic axonsin iris–ciliary bodies, presynaptic release-inhibiting mus-carinic receptors have been relatively well studied.Currently, five muscarinic receptor subtypes, M1–M5, aredistinguished (reviewed by Fuder and Muscholl 1995;Caulfield and Birdsall 1998; Alexander et al. 2001).Classical characterization by means of muscarinic receptorantagonists have shown that presynaptic muscarinicreceptors in iris–ciliary bodies are most similar to M2 inall species studied (Bognar et al. 1989, 1992; Dammann etal. 1989; Fuder et al. 1992; Jumblatt and Hackmiller1994). However, a clear subtype determination is oftenhindered by the lack of sufficiently subtype-selectivedrugs. Moreover, presynaptic modulation seems to bemediated in many cases by more than one subtype, leadingto mixed pharmacological properties (Altmann et al. 1999;Hein et al. 1999; Trendelenburg et al. 1999, 2001, 2003a,2003b).

A powerful new approach to receptor characterizationthat circumvents these difficulties is the use of transgenicanimals, which are almost exclusively mice. Experimentswith knockout mice have, for example, shown thatpresynaptic α2-autoreceptors in all sympathetic tissuesexamined are heterogeneous and represent a mixture of allthree known α2-subtypes, α2A–C (Altmann et al. 1999;Hein et al. 1999; Trendelenburg et al. 1999, 2001, 2003a).Similarly, muscarinic heteroreceptors on sympatheticaxons have been shown to be a mixture of M2-receptorsand non-M2-receptors (Trendelenburg et al. 2003b).However, such transgenic approaches were not applicablefor iris–ciliary bodies because 3H-noradrenaline releaseand its modulation have never been, respectively, studiedin the mouse.

The major aim of the present study was to establishmeasurement of sympathetic neurotransmitter release frommouse iris–ciliary bodies. Neurotransmitter release experi-ments were carried out with isolated preparations frommouse iris–ciliary bodies after labeling with 3H-noradren-aline. In addition, presynaptic muscarinic receptorsmediating inhibition of 3H-noradrenaline release wereidentified and classified. The muscarinic receptor agonistoxotremorine M and the antagonists ipratropium (high-affinity, non-selective) as well as methoctramine andpirenzepine that allow a limited distinction betweenmuscarinic receptor subtypes, were used as pharmacolo-gical tools: the rank order methoctramine > pirenzepine istypical for M2-receptors, whereas pirenzepine ≥ methoc-tramine is typical for all the other muscarinic receptors(see Trendelenburg et al. 2003b). Finally, it was studiedwhether endogenously released acetylcholine is able tomodulate noradrenaline release by administration ofipratropium in the presence of the acetylcholine esteraseinhibitor physostigmine.

Methods

Tissues and superfusion Male C57/Bl6 mice (25–45 g)were killed by cervical dislocation. From each mouse,both eyes were removed and iris–ciliary bodies weredissected. Tissues were preincubated in 1 ml medium (seebelow) containing 0.6 μM 3H-noradrenaline for 60 min at37°C and then placed in ten superfusion chambersbetween platinum wire electrodes, one iris–ciliary bodyper chamber, where they were superfused with 3H-noradrenaline-free medium at a rate of 1.2 ml/min−1.Successive 2-min samples of the superfusate werecollected from t=60 min onwards (t=0 min being thestart of superfusion). At the end of experiments, tissueswere dissolved and tritium was determined in superfusatesamples and tissues.

The superfusion medium contained (mM): NaCl 118,KCl 4.8, CaCl2 2.5, MgSO4 1.2, NaHCO3 25,KH2PO4 1.2, glucose 11, ascorbic acid 0.57, disodiumEDTA 0.03, and desipramine 0.001. The medium forpreincubation with 3H-noradrenaline contained no desi-pramine and only 0.2 mM CaCl2 (Limberger et al. 1992).

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Protocols Two slightly different protocols were used. Ineither protocol, there were five periods of electricalstimulation. Each stimulation period consisted of rectan-gular pulses of 1 ms width and 35 V/cm voltage dropbetween the electrodes of each chamber, yielding a currentstrength of 60 mA. The first stimulation period (180pulses/3 Hz) was delivered at t=30 min and was not usedfor determination of tritium overflow. Times and pulsepatterns of the subsequent stimulation periods (S1 to S4)depended on the protocol used. If not stated otherwise,experiments were done in the presence of α2-antagonists.A combination of phentolamine 1 μM (preferential mouseα2A-antagonist) and rauwolscine 1 μM (preferentialmouse α2B-antagonist and α2C-antagonist) was chosen toenable blockade of all three α2-adrenoceptor subtypeswhich were shown to operate as presynaptic autoreceptorsin mice (Trendelenburg et al. 2003a).

In protocol 1 (used in all experiments except thosedesigned to investigate the effect of endogenously releasedacetylcholine on presynaptic muscarinic heteroreceptors),the stimulation periods S1 to S4 were applied at t=64, 86,108, and 130 min and consisted of 36 pulses at 3 Hz.Drugs were either present throughout superfusion at afixed concentration or were introduced after S1, 12 minbefore S2, S3 or S4. In experiments designed to characterizepresynaptic muscarinic receptors, concentration–responsecurves for oxotremorine M were determined in the absenceand presence of muscarinic receptor antagonists. For thispurpose, oxotremorine M was introduced at increasingconcentrations after S1, 12 min before S2, S3 and S4.Antagonists were present throughout superfusion at afixed concentration.

In protocol 2 (used for experiments designed toinvestigate the effect of endogenously released acetylcho-line on presynaptic muscarinic heteroreceptors), the stim-ulation periods S1 to S4 were applied at t=64, 86, 126, and148 min. S1 and S3 consisted of 120 pulses/3 Hz and S2and S4 consisted of 600 pulses/5 Hz. All experiments weredone in the presence of the acetylcholine esterase inhibitorphysostigmine 1 μM to increase extracellular levels ofacetylcholine. Ipratropium 1 μM was introduced 30 minbefore S3.

Evaluation The outflow of tritium was calculated as afraction of the tritium content of the tissue at the onset ofthe respective collection period (fractional rate; min−1).The overflow elicited by electrical stimulation wascalculated as the difference “total tritium outflow duringand after stimulation” minus “basal outflow” and was thenexpressed as a percentage of the tritium content of thetissue at the time of stimulation (see Trendelenburg et al.1997).

For further evaluation, overflow ratios were calculated:Sn/S1 in experiments with identical stimulation patterns atS1 to S4, and Sn/Sn−2 in experiments with differentstimulation patterns (note that stimulation patterns at Snand Sn−2 were identical). Overflow ratios obtained in thepresence of drugs added after S1 or S2 were also calculatedas a percentage of the corresponding ratio in controls in

which no drug was added after S1 or S2. Effects of drugson basal tritium outflow were evaluated similarly (Tren-delenburg et al. 1997).

Concentration–response data for oxotremorine M givenalone were evaluated by sigmoid curve fitting (Eq. 25 ofWaud 1976). This yielded the Emax (maximal effect) ofoxotremorine M and its EC50 (concentration causing ahalf-maximal effect) in the absence of antagonist. In mostcases, sigmoid curves could not be fitted to concentration–response data for oxotremorine M in the presence ofantagonists. Therefore, for determination of apparentantagonist pKd values (negative logarithms of the apparentKd), EC50 values for oxotremorine M in the presence ofantagonist were interpolated from the nearest points of therespective concentration–response curves, assuming thatthe Emax of oxotremorine M had not changed; the pKd

value was then calculated from the increase in EC50 values(Eq. 4 of Furchgott 1972). The pKd values are apparentbecause only one antagonist concentration was used andthe competitive character of the interaction was notverified.

Results are expressed as arithmetic means±SEM(estimates±SE defined by Waud 1976, in the case ofEmax and EC50 values of oxotremorine M). Groups weretested for significant differences with ANOVA (usingSigmaStat). P<0.05 was taken as limit of statisticaldifference. n represents the number of iris–ciliary bodies.

Drugs Drugs were (−)-[ring-2,5,6-3H]-noradrenaline, spe-cific activity 49.7–52.4 Ci/mmol (Perkin Elmer, Geneva,Switzerland), desipramine HCl, ipratropium bromide,methoctramine 4 HCl, oxotremorine M, phentolaminemethanesulfonate, pirenzepine 2 HCl, rauwolscine HCl(Sigma-Aldrich, Buchs, Switzerland), physostigminehemisulfate and tetrodotoxin citrate (ANAWA, Wangen,Switzerland). Drugs were dissolved in distilled water.

Results

In the experiments described here, whole iris–ciliarybodies from mice were preincubated with 3H-noradrena-line to label vesicular noradrenaline pools. The release of3H-noradrenaline was elicited by electrical field stimula-tion.

Measurement of electrically evoked 3H-noradrenalinerelease

In this first step, experimental conditions for measurementof 3H-noradrenaline release were searched which areappropriate to investigate presynaptic inhibition. Iris–ciliary bodies were stimulated four times (S1–S4) by 36pulses/3 Hz. In the absence of α2-adrenoceptor antago-nists, electrical stimulation led to clear peaks of tritiumoverflow at S1–S4 (see Fig. 1A, B). Basal efflux before S1averaged to 0.0041±0.0002 min−1 (n=30). The magnitudeof the evoked overflow at S1 is shown in Table 1. In

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controls (no antagonists throughout), basal tritium effluxas well as the size of overflow peaks remainedapproximately constant from S1 to S4 as indicated by bn/b1 and Sn/S1 ratios close to unity (n=9; Fig. 1A, B).

Stimulation with relatively long pulse trains at lowfrequency like, for example, 36 pulses/3 Hz usually leadsto α2-autoinhibition of noradrenaline release in manytissues of various species (Trendelenburg et al. 1999,2000). These stimulation conditions also led to α2-autoinhibition in mouse iris–ciliary bodies as indicatedby the release-enhancing effect of α-adrenoceptor antago-nists: a combination of phentolamine 1 μM and rauwols-cine 1 μM given after S2 approximately doubled evokedtritium overflow at S3 and S4 (see Fig. 1A).

However, for studying presynaptic release-inhibitingheteroreceptors by exogenous agents, stimulation condi-tions with no or little α2-autoinhibition are optimal (seeStarke 1987; Schlicker and Göthert 1998). Thus, to blockα2-autoinhibition all subsequent experiments were con-ducted in the presence of a combination of phentolamine1 μM and rauwolscine 1 μM. Addition of phentolamine1 μM and rauwolscine 1 μM did not change basal tritiumoverflow before S1 (0.0042±0.0002 min−1; n=15), butapproximately doubled release peaks at S1 (compare lines1 and 2 in Table 1). In controls (phentolamine 1 μM andrauwolscine 1 μM throughout), evoked overflow of tritiumagain remained constant from S1 to S4, whereas basaloutflow slightly increased with time by maximally about10% (at b4).

Tetrodotoxin 0.3 μM when added to the superfusionmedium after S2 (Fig. 2) almost completely abolishedevoked overflow of tritium, indicating a neuronal origin ofthe 3H-noradrenaline release. This shows that the presentconditions are suitable to investigate 3H-noradrenalinerelease-inhibiting heteroreceptors in the mouse iris–ciliarybody by exogenous agents.

Identification and characterization of presynapticmuscarinic receptors inhibiting noradrenaline release

In this second step, 3H-noradrenaline release-inhibitingmuscarinic receptors were detected and characterized. Forthis purpose, mouse iris–ciliary bodies were stimulated asdescribed above (S1–S4: 36 pulses/3 Hz; phentolamine1 μM and rauwolscine 1 μM throughout). Concentration–response curves for the muscarinic receptor agonistoxotremorine M in the absence and presence of muscarinicreceptor antagonists were determined.

When given alone, oxotremorine M (10 nM–10 μM)reduced the evoked overflow of tritium in a concentration-dependent manner as shown in Figs. 1C, 3 (filled circles).The Emax of oxotremorine M (expressed as % inhibition ofstimulation-evoked tritium overflow compared to controlswithout oxotremorine M), obtained from logistic curvefitting, amounted to 51±9%, its EC50 value to 0.33±0.44 μM.

Fig. 1 Outflow of tritium frommouse iris–ciliary bodies: ef-fects of electrical stimulation, α-adrenoceptor antagonists andoxotremorine M. After preincu-bation with 3H-noradrenalinetissues were superfused andstimulated four times by 36pulses/3 Hz (S1–S4). In A and Bno antagonists were present andin C and D a combination ofphentolamine 1 μM and rau-wolscine 1 μM was presentthroughout superfusion. APhentolamine 1 μM and rau-wolscine 1 μM were added asindicated, B control (no antago-nists throughout), C oxotremor-ine M was added at increasingconcentrations as indicated, andD control (phentolamine 1 μMand rauwolscine 1 μM through-out). Each line represents means±SEM of 9–25 iris–ciliary bod-ies.

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To further characterize the receptors mediating thenoradrenaline release-inhibiting effect of oxotremorine M,the antagonists ipratropium (high-affinity, non-selective)as well as methoctramine and pirenzepine, which are ableto distinguish between some muscarinic receptor subtypes,were used. From the antagonists tested, only ipratropium3 nM, when present throughout superfusion, slightlychanged (about 20% reduction) the stimulation-evokedoverflow of tritium at S1. If iris–ciliary bodies weresuperfused in the presence of muscarinic receptorantagonists only (no oxotremorine M added after S1), Sn/S1 ratios again were close to unity (n=8–11 for eachantagonist). None of the muscarinic receptor ligands usedhad any significant effect on basal tritium outflow eventhough oxotremorine M at the highest concentration tested

(10 μM) tended to increase basal efflux (by about 20%;data not shown).

Ipratropium 3 nM and methoctramine 1 μM shifted theconcentration–response curve of oxotremorine M to theright (Fig. 3A, B), whereas pirenzepine 1 μM wasineffective (Fig. 3B). Apparent antagonist pKd valueswere calculated from the shifts (Fig. 3) leading to the rankorder of antagonist potency, ipratropium (pKd 9.7) >methoctramine (7.2) > pirenzepine (<6).

Inhibition of noradrenaline release by endogenousacetylcholine

In the final step, we investigated whether endogenouslyreleased acetylcholine can activate the 3H-noradrenalinerelease-inhibiting muscarinic receptors. To increase thechance that endogenous muscarinic inhibition of norad-renaline release develops, the levels of synaptic acetyl-choline were raised by using longer pulse trains. Thus,iris–ciliary bodies were stimulated by 120 pulses/3 Hz (S1and S3) and 600 pulses/5 Hz (S2 and S4). In the absence ofα2-antagonists, evoked overflow values (Table 1) were asexpected markedly bigger than those induced by 36pulses/3 Hz (120 pulses/3 Hz: ∼3-fold; 600 pulses/5 Hz:∼11-fold). These long pulse trains also led to considerableα2-autoinhibition, as indicated by the release-enhancingeffect of the α2-antagonists: a combination of phentola-mine 1 μM and rauwolscine 1 μM given after S2approximately doubled evoked tritium overflow to eachpulse pattern (n=7).

In all subsequent experiments α2-autoinhibition wasprevented by phentolamine 1 μM and rauwolscine 1 μM.Moreover, to further raise levels of synaptic acetylcholineexperiments were conducted in the presence of theacetylcholine esterase inhibitor physostigmine 1 μM.Basal efflux before S1 averaged to 0.0039±0.0002 min−1

(n=22). The magnitude of the evoked overflow at S1 andS2 is shown in Table 1. In controls (phentolamine 1 μM,rauwolscine 1 μM, and physostigmine 1 μM throughout),basal tritium efflux remained approximately constant fromS1 to S4 (Fig. 4B) as indicated by bn/b1 close to unity. Incontrast, evoked overflow to identical stimulation droppedwith time (Fig. 4B) as indicated by Sn/Sn−2 ratios belowunity: ∼0.60 (S3/S1) and ∼0.50 (S4/S2), respectively. Ifipratropium 1 μM was added after S2 evoked overflow to

Table 1 Electrically evoked tritium overflow from mouse iris–ciliary bodies

Drugs throughout Evoked tritium overflow (% of tissue tritium)

36 pulses/3 Hz (S1) 120 pulses/3 Hz (S1) 600 pulses/5 Hz (S2)

No antagonists 0.30±0.02 (n=31) 0.80±0.10 (n=14) 3.36±0.44 (n=14)Phentolamine 1 μM + rauwolscine 1 μM 0.64±0.04* (n=52) – –Phentolamine 1 μM + rauwolscine 1 μM + physostigmine 1 μM – 1.69±0.15* (n=22) 5.58±0.59* (n=22)

Iris–ciliary bodies were stimulated by either 36 pulses/3 Hz (S1), 120 pulses/3 Hz (S1) or 360 pulses/3 Hz (S2). As indicated either noantagonists, a combination of phentolamine 1 μM and rauwolscine 1 μM, or a combination of phentolamine 1 μM, rauwolscine 1 μM, andphysostigmine 1 μM were present throughout superfusion. Data are means±SEM from n ciliary bodiesSignificant difference from experiments without antagonists: *P<0.05

Fig. 2 Effect of tetrodotoxin on the evoked overflow of tritiumfrom mouse iris–ciliary bodies. After preincubation with 3H-noradrenaline tissues were superfused and stimulated electricallyfour times (S1–S4) by 36 pulses/3 Hz. A combination of phento-lamine 1 μM and rauwolscine 1 μM was present throughoutsuperfusion. Tetrodotoxin 0.3 μM was added from 12 min before S3onwards. Ordinate, evoked overflow of tritium, calculated from Sn/S1 ratios and expressed as a percentage of the corresponding control(phentolamine 1 μM and rauwolscine 1 μM throughout). Means±SEM from n=11 iris–ciliary bodies. Significant differences fromcorresponding control (phentolamine 1 μM and rauwolscine 1 μMthroughout): *P<0.05

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each pulse pattern was enhanced by about 100% (Figs. 4,5).

Discussion

To our knowledge, we describe here for the first timemeasurement of 3H-noradrenaline release from mouseiris–ciliary bodies. In addition, existence of release-inhibiting muscarinic heteroreceptors on sympatheticaxons of mouse iris–ciliary body, most similar to M2, isreported. Their contribution to the interplay between theparasympathetic and sympathetic nervous system was alsostudied.

Measurement of electrically evoked 3H-noradrenalinerelease

3H-noradrenaline release from iris–ciliary body prepara-tions and its modulation by presynaptic receptors havebeen studied for more than 30 years in various speciesincluding man, but not in the mouse (see Introductionsection). However, new molecular genetic approaches toreceptor classification have moved the species mouse tothe center of interest. The use of transgenic animals, whichare almost exclusively mice, has been shown to circum-vent the difficulties associated with limited subtype-selective pharmacological tools (Altmann et al. 1999;Hein et al. 1999; Trendelenburg et al. 1999, 2001, 2003a,2003b; see also Hein 2001; Wess 2003).

The present study establishes measurement of norad-renaline release from mouse iris–ciliary bodies afterpreincubation with 3H-noradrenaline. It is well knownthat experimental conditions leading to noradrenalinerelease with little or no α2-autoinhibition are optimal tostudy presynaptic sympathetic modulation by exogenousligands (see Starke 1987; Schlicker and Göthert 1998).Usually stimulation with short, high-frequency pulse trains(e.g., 20 pulses/50 Hz) led to such optimal release.Unfortunately, 3H-noradrenaline release was not reliablymeasurable if mouse iris–ciliary bodies were stimulatedwith such pulse trains (data not shown). In contrast,stimulation with relatively long, low-frequency pulsetrains (i.e., 36 pulses/3 Hz) led to measurable andreproducible release peaks. As expected for such stimu-lation conditions, marked α2-autoinhibition occurred and,thus, to study presynaptic muscarinic inhibition, α2-autoinhibition was prevented by addition of α2-antagoniststo the superfusion medium. Another important prerequisitefor studying presynaptic modulation is to choose condi-tions leading to “quasi-physiological” action potential-induced transmitter release. Accordingly, the almostcomplete inhibition of electrically evoked tritium overflowby tetrodotoxin 0.3 μM confirms the neuronal origin of3H-noradrenaline release. Thus, the experimental condi-tions described here (i.e., stimulation with 36 pulses/3 Hzin the presence of α2-blockade) are suitable for investigat-ing presynaptic sympathetic modulation in mouse iris–ciliary bodies.

Fig. 3 Interaction of muscarinic antagonists with oxotremorine Mon the evoked overflow of tritium from mouse iris–ciliary bodies.After preincubation with 3H-noradrenaline tissues were superfusedin the presence of a combination of phentolamine 1 μM andrauwolscine 1 μM and stimulated electrically four times by 36pulses/3 Hz (S1–S4). Oxotremorine M was added at increasingconcentrations (abscissae) before S2–S4. Oxotremorine M was giveneither alone or combined with antagonists, which were presentthroughout superfusion. In A interaction with ipratropium 3 nM and

in B interactions with methoctramine 1 μM and pirenzepine 1 μMare shown. Ordinates, evoked overflow of tritium, calculated fromSn/S1 ratios and expressed as a percentage of the correspondingcontrol (phentolamine 1 μM and rauwolscine 1 μM throughout).Means±SEM from n=6–20 iris–ciliary bodies. Significant differ-ences from corresponding control (phentolamine 1 μM andrauwolscine 1 μM throughout): *P<0.05. Significant differencesfrom the effect of oxotremorine M in the absence of antagonist:#P<0.05

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Identification and characterization of muscarinicreceptors inhibiting noradrenaline release

It was evident from previous studies that postganglionicsympathetic axons in the iris–ciliary body possess pre-synaptic muscarinic receptors inhibiting noradrenalinerelease. Such presynaptic muscarinic receptors have beendetected in iris–ciliary bodies from rats, guinea pigs,rabbits as well as in humans. Moreover, classical receptorcharacterization by means of muscarinic receptor antago-nists has shown that these receptors are most similar to M2

in all species examined (Jumblatt and North 1988; Bognaret al. 1988, 1989, 1992; Dammann et al. 1989; Fuder et al.1989; Jumblatt and Hackmiller 1994). With a similarapproach, we have identified in the present study presyn-aptic muscarinic receptors inhibiting noradrenaline releasein the iris–ciliary body of mice. Their existence is evidentfrom both the release-inhibiting effect of oxotremorine Mand the counteracting effects of muscarinic receptorantagonists. The rank order of antagonist potenciesobserved, ipratropium > methoctramine > pirenzepine,

which is the typical M2 order (see Trendelenburg et al.2003b), clearly shows that the pharmacological propertiesof presynaptic muscarinic receptors in mouse iris–ciliarybodies are, in accord with findings in other species, mostsimilar to M2. This indicates that presynaptic release-inhibiting muscarinic receptors in the iris–ciliary bodyseem to be predominantly M2, across mammalian species.

At a first glance, this M2 diagnosis is also in accord withthe identification of M2 muscarinic heteroreceptors onsympathetic axons in mouse atria, urinary bladder, and vasdeferens (Trendelenburg et al. 2003b) as well as in mousecultured sympathetic neurons (Göbel et al. 2000). How-ever, experiments with muscarinic receptor knockout micehave also shown that presynaptic muscarinic receptors onsympathetic axons are not homogenous. In all tissuesexamined, they represent a mixture of M2-receptors andnon-M2-receptors (Trendelenburg et al. 2003b). Thiscomplexity remained undetected in classical functionalstudies in wild-type mice using different muscarinicreceptor antagonists and, thus, is likely to occur in theiris–ciliary body as well. To clarify the uncertainty ifadditional muscarinic receptors subtypes contribute to thepresynaptic muscarinic inhibition in mouse iris–ciliarybody, respective knockout experiments would be of greatinterest.

Fig. 4 Outflow of tritium from mouse iris–ciliary bodies: effects ofelectrical stimulation and ipratropium. After preincubation with 3H-noradrenaline tissues were superfused and stimulated electricallyfour times (S1 to S4). S1 and S3 consisted of 120 pulses/3 Hz and S2and S4 of 600 pulses/5 Hz. A combination of phentolamine 1 μMand rauwolscine 1 μM as well as physostigmine 1 μM were presentthroughout superfusion. A Ipratropium 1 μM added as indicated,and B controls (phentolamine 1 μM, rauwolscine 1 μM, andphysostigmine 1 μM throughout). Each line represents means ±SEM of 11 iris–ciliary bodies.

Fig. 5 Effect of ipratropium on the evoked overflow of tritium frommouse iris–ciliary bodies. After preincubation with 3H-noradrena-line tissues were superfused and stimulated electrically four times(S1–S4). S1 and S3 consisted of 120 pulses/3 Hz and S2 and S4 of 600pulses/5 Hz. A combination of phentolamine 1 μM and rauwolscine1 μM as well as physostigmine 1 μM were present throughoutsuperfusion. Ordinate, evoked overflow of tritium calculated fromSn/Sn−2 ratios and expressed as a percentage of the correspondingcontrol (phentolamine 1 μM, rauwolscine 1 μM, and physostigmine1 μM throughout). Means±SEM from n=11 iris–ciliary bodies.Significant differences from corresponding control (phentolamine1 μM, rauwolscine 1 μM, and physostigmine 1 μM throughout):*P<0.05

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Activation of muscarinic heteroreceptors byendogenous acetylcholine

Various studies have shown that endogenously releasedacetylcholine is able to trigger, at least under certainexperimental conditions, muscarinic receptor-mediatedinhibition of sympathetic transmitter release suggestingthat presynaptic muscarinic receptors might be physiolo-gically relevant. In accordance with this suggestion,muscarinic receptor antagonists were reported to increaseelectrically evoked 3H-noradrenaline release in severalpreparations such as mouse atria and urinary bladder, raturinary bladder as well as rabbit and human iris–ciliarybody (Bognar et al. 1988; Somogyi and de Groat 1990;Costa and Majewski 1991; Jumblatt et al. 1993; Trende-lenburg et al. 2003b).

We demonstrate here that a similar inhibition ofnoradrenaline release by endogenous acetylcholine occursin mouse iris–ciliary bodies. When as in previous studiesrelatively long pulse trains, 120 pulses/3 Hz (as for mouseatria and bladder) and 600 pulses/5 Hz (as for human iris–ciliary body) were applied and physostigmine 1 μM (as formouse atria and bladder) was added to the superfusionmedium to enhance the chance for the development ofmuscarinic inhibition, ipratropium facilitated 3H-norad-renaline release. Presynaptic muscarinic heteroreceptorson sympathetic axons in the mouse iris–ciliary body seemto be under tonic innervation and, thus, might be ofphysiological relevance.

In conclusion, electrically evoked 3H-noradrenalinerelease is reliably measurable in mouse iris–ciliary bodies.Like in other species, this 3H-noradrenaline release isinhibited by presynaptic muscarinic receptors whichappear to be predominantly M2. Moreover, presynapticmuscarinic receptors can be activated by endogenousacetylcholine and may play a role in the interplay betweenthe sympathetic and parasympathetic nervous system inthe mouse iris–ciliary body. Similar experiments withreceptor knockout mice may contribute to a betterunderstanding of presynaptic molecular mechanisms inthe iris–ciliary body.

References

Alexander SPH, Mathie A, Peters JA (2001) TiPS receptor and ionchannel nomenclature supplement 2001. Elsevier, Cambridge

Altmann JD, Trendelenburg AU, MacMillan L, Bernstein D,Limbird L, Starke K, Kobilka BK, Hein L (1999) Abnormalregulation of the sympathetic nervous system in α2A-adrenergicreceptor knockout mice. Mol Pharmacol 56:154–161

Boehm S, Kubista H (2002) Fine tuning of sympathetic transmitterrelease via ionotropic and metabotropic presynaptic receptors.Pharmacol Rev 54:43–99

Bognar IT, Pallas S, Fuder H, Muscholl E (1988) Muscarinicinhibition of [3H]-noradrenaline release on rabbit iris in vitro:effects of stimulation conditions on intrinsic activity ofmethacholine and pilocarpine. Br J Pharmacol 94:890–900

Bognar IT, Baumann B, Dammann F, Knöll B, Meincke M, Pallas S,Fuder H (1989) M2 muscarinic receptors on the iris sphinctermuscle differ from those on iris noradrenergic nerves. Eur JPharmacol 163:263–274

Bognar IT, Altes U, Beinhauer C, Kessler I, Fuder H (1992) Amuscarinic receptor different from the M1, M2, M3 and M4subtypes mediates the contraction of the rabbit iris sphincter.Naunyn-Schmiedebergs Arch Pharmacol 345:611–618

Burke JA, Potter DE (1986) Ocular effects of a relatively selectivealpha 2 agonist (UK-14,304-18) in cats, rabbits and monkeys.Curr Eye Res 5:665–676

Caulfield MP, Birdsall NJM (1998) International Union of Pharma-cology. XVII. Classification of muscarinic acetylcholinereceptors. Pharmacol Rev 50:279–290

Chu E, Chu TC, Potter DE (1999) Potential sites of action of TNPA:a dopamine-2 receptor agonist. Exp Eye Res 69:611–616

Costa M, Majewski H (1991) Evidence for facilitatory andinhibitory muscarinic receptors on postganglionic sympatheticnerves in mouse isolated atria. Br J Pharmacol 102:855–860

Crosson CE, Gray T (1997) Response to prejunctional adenosinereceptors is dependent on stimulus frequency. Curr Eye Res16:359–364

Dammann F, Fuder H, Giachetti A, Giraldo E, Kilbinger H,Micheletti R (1989) AF-DX 116 differentiates betweenprejunctional muscarine receptors located on noradrenergicand cholinergic nerves. Naunyn-Schmiedebergs Arch Pharma-col 339:268–271

Drago F, Gorgone G, Spina F, Panissidi G, Dal Bello A, Moro F,Scapagnini U (1980) Opiate receptors in the rabbit iris.Naunyn-Schmiedebergs Arch Pharmacol 315:1–4

Farnebo LO, Hamberger B (1970) Effects of desipramine, phento-lamine and phenoxybenzamine on the release of noradrenalinefrom isolated tissues. J Pharm Pharamcol 22:855–857

Farnebo LO, Hamberger B (1971) Drug-induced changes in therelease of [3H]-noradrenaline from field stimulated rat iris. Br JPharmacol 43:97–106

Fuder H (1994) Functional consequences of prejunctional receptoractivation or blockade in the iris. J Ocul Pharmacol 10:109–123

Fuder H, Muscholl E (1995) Heteroreceptor-mediated modulation ofnoradrenaline and acetylcholine release from peripheral nerves.Rev Physiol Biochem Pharmacol 126:265–412

Fuder H, Muth U (1993) ATP and endogenous agonists inhibitevoked [3H]-noradrenaline release in rat iris via A1 and P2Y-likepurinoreceptors. Naunyn-Schmiedebergs Arch Pharmacol348:352–357

Fuder H, Schöpf J, Unckell J, Wesner MT, Melchiorre C, Tacke R,Mutschler E, Lambrecht G (1989) Different muscarinereceptors mediate the prejunctional inhibition of [3H]-norad-renaline release in rat or guinea-pig iris and the contraction ofthe rabbit iris sphincter muscle. Naunyn-Schmiedebergs ArchPharmacol 345:611–618

Fuder H, Brink A, Meincke M, Tauber U (1992) Purinoceptor-mediated modulation by endogenous and exogenous agonistsof stimulation-evoked [3H]-noradrenaline release on rat iris.Naunyn-Schmiedebergs Arch Pharmacol 345:417–423

Furchgott RF (1972) The classification of adrenoceptors (adrenergicreceptors). An evaluation from the standpoint of receptortheory. In: Blaschko H, Muscholl E (eds) Catecholamines.Handbook of experimental pharmacology, vol 33. Springer,Berlin Heidelberg New York, pp 283–335

Göbel I, Trendelenburg AU, Cox SL, Meyer A, Starke K (2000)Electrically evoked release of [3H]noradrenaline from mousecultured sympathetic neurons: release-modulating heterorecep-tors. J Neurochem 75:2087–2094

Harris LC, Awe SO, Opere CA, LeDay AM, Ohia SE (2002)Pharmacology of serotonin receptors modulating electrically-induced [3H]-noradrenaline release from isolated mammalianiris–ciliary bodies. J Ocul Pharmacol Ther 18:339–348

Hein L (2001) Transgenic models of α2-adrenergic receptor subtypefunction. Rev Physiol Biochem Pharmacol 142:161–185

Hein L, Altman JD, Kobilka BK (1999) Two functionally distinctα2-adrenergic receptors regulate sympathetic neurotransmis-sion. Nature 402:181–184

Jumblatt JE (1994) Prejunctional alpha 2-adrenoceptors andadenylyl cyclase regulation in the rabbit iris–ciliary body. JOcul Pharmacol 10:617–621

312

Jumblatt JE, Hackmiller RC (1990) Potentiation of norepinephrinesecretion by angiotensin II in the isolated rabbit iris–ciliarybody. Curr Eye Res 9:169–176

Jumblatt JE, Hackmiller RC (1994) M2-type muscarinic receptorsmediate prejunctional inhibition of norepinephrine release inthe human iris–ciliary body. Exp Eye Res 58:175–180

Jumblatt JE, North GT (1988) Cholinergic inhibition of adrenergicneurosecretion in the rabbit iris–ciliary body. Invest Ophthal-mol Vis Sci 29:615–620

Jumblatt JE, Liu JG, North GT (1987) Alpha-2 adrenergicmodulation of norepinephrine secretion in the perfused rabbitiris–ciliary body. Curr Eye Res 6:767–777

Jumblatt JE, Ohia SE, Hackmiller RC (1993) Prejunctional mod-ulation of norepinephrine release in the human iris–ciliarybody. Invest Ophthalmol Vis Sci 34:2790–2793

Koss MC, Hey JA (1993) Prejunctional inhibition of sympatheti-cally evoked pupillary dilatation in cats by activation ofhistamine H3 receptors. Naunyn-Schmiedebergs Arch Pharma-col 348:141–145

Kulkarni KH, LeDay AM, Opere CA, Ohia SE (2002) Pharmacol-ogy of prejunctional histamine receptors on sympathetic nervesin isolated mammalian irides. Invest Ophthalmol Vis Sci 43:e-abstract 1975

Langer SZ (1997) 25 years since the discovery of presynapticreceptors: present knowledge and future perspectives. TrendsPharmacol Sci 18:95–99

Limberger N, Trendelenburg AU, Starke K (1992) Pharmacologicalcharacterization of presynaptic α2-autoreceptors in rat submax-illary gland and heart atrium. Br J Pharmacol 107:246–255

Neufeld AH, Page ED (1975) Regulation of adrenergic neuromus-cular transmission in the rabbit iris. Exp Eye Res 20:549–561

Nietgen GW, Schmidt J, Hesse L, Hönemann CW, Durieux ME(1999) Muscarinic receptor functioning and distribution in theeye: molecular basis and implications for clinical diagnosis andtherapy. Eye 13:285–300

Ogidigben M, Chu TC, Potter DE (1993) Ocular hypotensive actionof a dopaminergic (DA2) agonist, 2,10,11-trihydroxy-N-n-propylnoraporphine. J Pharmacol Exp Ther 267:822–827

Ohia SE, Jumblatt JE (1990a) Prejunctional inhibitory effects ofprostanoids on sympathetic neurotransmission in the rabbit iris–ciliary body. J Pharmacol Exp Ther 255:11–16

Ohia SE, Jumblatt JE (1990b) Inhibitory effects of neuropeptide Yon sympathetic neurotransmission in the rabbit iris–ciliarybody. Neurochem Res 15:251–256

Ohia SE, Jumblatt JE (1991) Prejunctional prostaglandin receptorsin the human iris–ciliary body. Curr Eye Res 10:967–975

Ohia SE, Jumblatt JE (1993) Prejunctional receptors and secondmessengers for angiotensin II in the rabbit iris–ciliary body.Exp Eye Res 57:419–425

Opere CA, Ohia SE (1998) Prejunctional alpha2-adrenoceptors andperoxide-induced potentiation of norepinephrine release fromthe bovine iris. Neurochem Res 23:1093–1098

Russel KR, Potter DE (2001) Biphasic alterations of cAMP andinhibition of norepinephrine release in iris–ciliary body bybremazocine. J Pharmacol Exp Ther 298:941–946

Schlicker E, Göthert M (1998) Interactions between the presynapticα2-autoreceptor and presynaptic inhibitory heteroreceptors onnoradrenergic neurones. Brain Res Bull 47:129–132

Shara M, Kulkarni KH, Ohia SE (2002) Effect of cannabinoids onsympathetic neurotransmission in isolated bovine irides. InvestOphthalmol Vis Sci 43:e-abstract 3273

Somogyi GT, de Groat WC (1990) Modulation of the release of [3H]norepinephrine from the base and body of the rat urinarybladder by endogenous adrenergic and cholinergic mechan-isms. J Pharmacol Exp Ther 255:204–210

Starke K (1987) Presynaptic α-autoreceptors. Rev Physiol BiochemPharmacol 107:73–146

Trendelenburg AU, Sutej I, Wahl CA, Molderings GJ, Rump LC,Starke K (1997) A re-investigation of questionable subclassi-fications of presynaptic α2-autoreceptors: rat vena cava, ratatria, human kidney and guinea-pig urethra. Naunyn-Schmie-debergs Arch Pharmacol 356:721–737

Trendelenburg AU, Hein L, Gaiser EG, Starke K (1999)Occurrence, pharmacology and function of presynaptic α2-autoreceptors in α2A/D-adrenoceptor-deficient mice. Naunyn-Schmiedebergs Arch Pharmacol 360:540–551

Trendelenburg AU, Cox SL, Schelb V, Klebroff W, Khairallah L,Starke K (2000) Modulation of 3H-noradrenaline release bypresynaptic opioid, cannabinoid and bradykinin receptors andβ-adrenoceptors in mouse tissues. Br J Pharmacol 130:321–330

Trendelenburg AU, Klebroff W, Hein L, Starke K (2001) A study ofpresynaptic α2-autoreceptors in α2A/D-, α2B-, α2C-adrenocep-tor-deficient mice. Naunyn-Schmiedebergs Arch Pharmacol364:117–130

Trendelenburg AU, Philipp M, Meyer A, Klebroff W, Hein L, StarkeK (2003a) All three α2-adrenoceptor types serve as auto-receptors in postganglionic sympathetic neurons. Naunyn-Schmiedebergs Arch Pharmacol 368:504–512

Trendelenburg AU, Gomeza J, Klebroff W, Zhou H, Wess J (2003b)Heterogeneity of presynaptic muscarinic receptors mediatinginhibition of sympathetic transmitter release: a study with M2-and M4-receptor-deficient mice. Br J Pharmacol 138:469–480

Waud DR (1976) Analysis of dose–response relationships. In:Narahshi T, Bianchi CP (eds) Advances in general and cellularpharmacology, vol 1. Plenum, New York, pp 145–178

Wess J (2003) Novel insights into muscarinic acetylcholine receptorfunction using gene targeting technology. Trends Pharmacol Sci24:414–420

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