Somatic and prejunctional nicotinic receptors in cultured rat sympathetic neurones show different agonist profiles

Transcription

1 8791 Journal of Physiology (1999), 516.3, pp Somatic and prejunctional nicotinic receptors in cultured rat sympathetic neurones show different agonist profiles D. Kristufek, E. Stocker, S. Boehm and S. Huck Department of Neuropharmacology, University of Vienna, W ahringerstra³e 13A, A_1090 Vienna, Austria (Received 29 September 1998; accepted after revision 3 February 1999) 1. The release of [ÅH]-noradrenaline ([ÅH]-NA) in response to nicotinic acetylcholine receptor (nachr) agonists was compared with agonist-induced currents in cultured rat superior cervical ganglion (SCG) neurones. 2. [ÅH]-NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd to block voltage-gated Na or Ca channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX- or Cd -insensitive release) to pharmacologically characterize nachrs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nachrs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine>ach,andwithec50 values ranging from 22 ìò (DMPP) to 110 ìò (ACh). 3. [ÅH]-NA release in response to low concentrations of nachr agonists was fully inhibited by the presence of either TTX or Cd (named TTX- or Cd -sensitive release). TTX-sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine nicotine ACh, which due to its similarity to TTX-insensitive release indicates that it might also be triggered by prejunctional-type nachrs. The EC50 values for TTX (Cd )-sensitive release were less than 10 ìò for all four agonists. 4. By contrast to transmitter release, somatic nachrs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine DMPP > ACh. EC50 values for the induction of currents exceeded 20 ìò for all four agonists. 5. The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. á-bungarotoxin (á-butx) was, on the other hand, without significant effect on nicotine-induced TTX-insensitive release. The competitive antagonist dihydro-âerythroidine (DHâE) caused rightward shifts of the dose response curves for both TTXsensitive and TTX-insensitive transmitter release as well as for currents in response to nicotine, with paµ values ranging from 4 03 to Due to clear differences in the pharmacology of agonists we propose that nachrs of distinct subunit composition are differentially targeted to somatic or axonal domains. Fast cholinergic transmission through sympathetic ganglia is mediated by the activation of somatodendritic nicotinic acetylcholine (ACh) receptors (nachrs) located within the ganglia. However, it has long been known that these neurones, in addition, have prejunctional nachrs on their nerve endings in target organs, where nicotinic agonists cause the release of noradrenaline (NA) (Starke, 1977). In the peripheral nervous system (PNS), the biological significance of these prejunctional nachrs is still a matter of debate (Starke, 1977; Fuder & Muscholl, 1995). This is in contrast to the central nervous system (CNS), where presynaptic nachrs may have surpassed their postsynaptic counterparts (Role & Berg, 1996; Wonnacott, 1997). Studies in vegetative ganglia have contributed a major share to the understanding of the function and the molecular biology of neuronal nachrs (for reviews see Sargent, 1993; McGehee & Role, 1995b). Rat superior cervical ganglion (SCG) neurones have high levels of á3 and â4 subunit mrna, but they also show á7, â2, á5 and á4.1 mrna (Mandelzys et al. 1994; Rust et al. 1994; Klimaschewski et al. 1994; DeKoninck & Cooper, 1995). The many possible combinations raise the possibility that functionally distinct

2 740 D. Kristufek, E. Stocker, S. Boehm and S. Huck J. Physiol nachrs might be directed to specific subcellular sites. Morphological studies with the combined use of neuronal bungarotoxin and á-bungarotoxin (á_butx) suggested ultrastructural heterogeneity of nachrs at the surface membrane of rat SCG neurones (Loring et al. 1988). We have previously shown that ACh induces the release of [ÅH]-noradrenaline ([ÅH]-NA) in cultured rat sympathetic neurones even when voltage-activated Ca channels (VACCs) have been blocked by Cd (Boehm & Huck, 1995). Given the Ca dependence of this phenomenon (Boehm & Huck, 1995), our observations implied a prejunctional mechanism of Ca influx independent of VACCs, presumably directly through nachrs (Role & Berg, 1996; Wonnacott, 1997). We have now used this component of transmitter release to define properties of prejunctional nachrs by means of pharmacological tools. The results from these experiments were compared with pharmacological data obtained by whole-cell patch clamp recordings, since currents elicited by nicotinic agonists are expected to reflect the properties of nachrs at somatodendritic domains. Patch clamp as well as transmitter release experiments included not only dose response curves from pooled observations, but also low-concentration potency ratios of agonists in the same experimental setting (Covernton et al. 1994). The observed clear differences in the rank order of potencies of agonists led us to conclude that the prejunctional receptors at varicosities are pharmacologically distinct from those at the soma, consistent with differential targeting of nachrs of distinct subunit composition. METHODS Cell culture Superior cervical ganglia (SCG) were dissected from 2 6 day old Sprague Dawley rat pups killed by decapitation, and were dissociated as described previously (Boehm & Huck, 1995). Briefly, ganglia were freed from adhering connective tissue and blood vessels, cut into three to four pieces, and incubated in collagenase (1 5 mg ml ; Sigma, C_9891) and dispase (3 0 mg ml ; Boehringer Mannheim, ) for 20 min at 36 5 C. Subsequently, the ganglia were trypsinized (0 25 % trypsin in Tyrode solution; Worthington, 3703) for 15 min at 36 5 C and dissociated by trituration. Dissociated cells were resuspended in serum-free culture medium and plated either onto 5 mm discs (punched out of tissue culture dishes; Nunc, ) coated with collagen (Biomedical Technologies, BT_274) for release experiments, or onto poly-lornithine (100 mg l HµO; Sigma, P_3655)-treated tissue culture dishes for patch clamp recordings. Cells were seeded into glass rings of 6 mm ( cells for release experiments) or 8 mm diameter ( cells for patch clamp) in order to confine the cell suspension to the discs or to the centre of the dishes. The culture medium consisted of Dulbecco s modified Eagle s medium (GibcoBRL, 81885_023), supplemented with 2 2 g l glucose, 10 mg l insulin (Sigma, I_5500), IU l penicillin, 25 mg l streptomycin (GibcoBRL, 15140_106) and 10 ìg l nerve growth factor (Peninsula Laboratories, IP_9020). The glass rings were removed after 90 min of incubation, and heat-inactivated fetal calf serum (GibcoBRL, 10108_157) was added to a final concentration of 5 %. The cells were kept in vitro in 5 % COµ, 95 % air and at 36 5 C for 3 6 days prior to transmitter release or patch clamp experiments. Cultures keptformorethan4dayswerefedafter2or3daysin vitro by replacing half of the culture medium. Under the culture conditions used for electrophysiology, cells more closely resembled dendritedeficient than dendrite-bearing neurones (Lein et al. 1995), and we therefore refer to our own results on receptor-mediated currents by using the term somatic rather than somatodendritic nachrs. By adopting this terminology we do, however, not exclude that dendritic nachrs contribute to agonist-induced currents. [ÅH]-Noradrenaline uptake and superfusion The techniques of labelling cultures of rat sympathetic neurones with [ÅH]-noradrenaline (NA) and subsequent superfusion have previously been described in detail (Boehm & Huck, 1995). The cultures were incubated in 0 03 ìò [ÅH]-NA in culture medium containing 1 mò ascorbic acid for 60 min. Thereafter, culture discs were transferred to small chambers and superfused with a buffer containing (mmol l ): NaCl, 120; KCl, 3 0; CaClµ, 2 0; MgClµ, 2 0; glucose, 20; Hepes, 10; fumaric acid, 0 5; sodium pyruvate, 5 0; and ascorbic acid, 0 57; adjusted to ph 7 4 with NaOH, 25 C, at a superfusion rate of 1 1 ml min. The radioactivity released into the superfusate of such cultures has been shown to consist primarily of authentic [ÅH]-NA (Schwartz & Malik, 1993). Subsequent to a 60 min washout period, the collection of 4 min superfusate fractions was started. ÅH outflow was induced either by electrical field stimulation (100 monophasic rectangular pulses delivered at 10 Hz, 0 5 ms, 50 V cm, 40 ma), or by adding nicotinic agonists to the superfusion medium for 15 s. Cultures were challenged with a first stimulus (S1) 12 min after the washout period (i.e. after 72 min of superfusion) and to a maximum of four additional stimuli at 20 min intervals. Concentration response relationships for agonists were established by a total of four stimuli with increasing concentrations of the agent. Unless otherwise noted, substances such as nicotinic antagonists or TTX were present either throughout the collection period or added 8 min before and present during Sµ. At the end of an experiment, the remaining radioactivity in the cultures was extracted by perchloric acid and sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting. Calculation of basal and stimulation-evoked tritium outflow The basal rate of ÅH outflow was obtained by dividing the radioactivity of a 4 min fraction by the total radioactivity of cultures at the beginning of the corresponding 4 min collection period. Basal outflow before S1 was termed L1. Stimulation-evoked outflow was calculated as the difference between the total ÅH outflow during and after stimulation on the one hand, and the estimated basal outflow on the other hand, assuming that basal release follows a linear decline. The (stimulus-induced) difference was expressed as a percentage of the total radioactivity in the cultures at the beginning of the respective stimulation (S %). The effects of drugs added between the two stimuli were evaluated by calculatingratiosoftheoutflowevokedbysµands1(sµï S1). Electrophysiology Electrophysiological recordings from neurones were performed at room temperature (20 24 C) by means of the perforated patch clamp technique (Rae et al. 1991). We used LÏM-EPC_7 (List Biomedical) or Axopatch 200A (Axon Instruments) patch clamp amplifiers and pclamp hardware and software (Axon Instruments). Substances were applied by a DAD_12 superfusion system (Adams & List). Unless otherwise noted, agonists were applied for 1 s, and antagonists were added to the superfusion buffer 10 s before and during application of agonists. The bathing solution contained (mò): NaCl, 120; KCl, 3 0; CaClµ, 2 0; MgClµ, 2 0; glucose, 20;

3 J. Physiol Somatic and prejunctional nicotinic receptors 741 Hepes, 10; and TTX, 0 001; adjusted to ph 7 3 with NaOH. The internal (pipette) solution contained (mò): KµSOÚ, 75; KCl, 55; MgClµ, 8 0; and Hepes, 10; adjusted to ph 7 3 with KOH. Access to cells was achieved by including 200 ìg ml amphotericin B (Sigma, A_4888) in the internal solution and by otherwise following the directions provided by Rae et al. (1991). Recordings thus obtained were stable for hours without signs of rundown which often accompanies agonist-induced currents recorded by means of the conventional whole-cell patch clamp technique. Statistics All data represent arithmetic means ± s.e.m. Full concentration response curves for agonists were fitted by unweighted non-linear regression to the logistic equation: Ex = Emaxx p Ï(x p p +EC50 ), (1) where Ex is the response; x the arithmetic dose; Emax the maximal response; p a slope factor; and ECÛÑ the dose that gives a halfmaximal response. Computation was done by using either SigmaPlot (Jandel Corporation) or the ALLFIT program (DeLean et al. 1978). The ALLFIT program calculates parameter estimates for EC50 values, p, and Emax, as well as appropriate standard errors and leaves the option to fit curves by weighted as well as by unweighted non-linear regression. Curve fits to the logistic equation were carried out with the intention of providing estimates for EC50 values of agonists and for curve shifts caused by competitive antagonists. Estimates for the slope (p), which is numerically identical to the Hill coefficient (DeLean et al. 1978), are provided primarily for the purposes of comparison with the literature, since slopes in a complex system like agonist-induced transmitter release will not only be determined by ligand receptor interaction, but also by factors such as receptor desensitization (Covernton et al. 1994) and mechanisms downstream of ligand receptor interaction (e.g. Ca -mediated exocytosis). Low-concentration potency ratios were determined according to Covernton et al. (1994). Hence, curves from a single cell or a single release experiment were fitted simultaneously by non-linear regression with logistic equations that were constrained to be parallel (i.e. with a shared slope parameter, p). The maximum response was fixed to a value inferred from full-range concentration response curves of previous experiments. Adequacy of the fits was judged by eye. IC50 values were defined as the concentration of an antagonist that caused 50 % inhibition of the response to a fixed concentration of an agonist. Mechanisms of competitive antagonism were judged by means of the Schild regression (Arunlakshana & Schild, 1959): log(x 1)=logKé npax, (2) where pax is the negative logarithm of the concentration of the inhibitor, x is the shift of the dose response curve (dose ratio) caused by its presence, Ké is the dissociation constant of the inhibitor, and n is the slope factor. The regression line fitted to the data intersects the abscissa at a point corresponding to paµ which in the case of n = 1 corresponds to Ké (Arunlakshana & Schild, 1959). The significance of differences for paired and unpaired observations was evaluated by the signed-rank tests according to Wilcoxon or Mann Whitney, respectively. Materials Drugs were obtained from the following sources: ( )-[ring_2,5,6- ÅH]-noradrenaline (56 9 Ci mmol ; NET678) from NEN; acetylcholine chloride (ACh; A_6625), ( )-noradrenaline hydrochloride (NA; A_7381), 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; D_5891), cytisine (C_2899), CdClµ (C_5081) and mecamylamine (M_9020) from Sigma; dihydro-â-erythroidine (DHâE; D_149) from RBI; and TTX (L8503) and á_bungarotoxin (á_butx; L8115) from Latoxan. Ordinary chemicals were from Merck (analytical grade). Drugs were stored as stock solutions in HµO at 20 C and diluted to the final concentrations in superfusion buffer for release experiments or in bathing solution for patch clamp recordings. RESULTS Two components of nicotine-induced transmitter release Two primary mechanisms may account for transmitter release in response to nachr activation. First, nicotinic agonists depolarize neurones to the threshold of voltagegated Na channels. The ensuing action potential then causes Ca entry via VACCs and Ca -dependent exocytosis. Second, Ca -dependent exocytosis occurs independently of both voltage-gated Na and Ca channels by Ca entry directly via nachrs located at presynaptic domains (Role & Berg, 1996; Wonnacott, 1997). Hence, the obvious tools to discriminate between these two mechanisms are TTX and Cd, which would block voltage-gated Na and Ca channels, respectively. We first determined the concentrations of TTX and Cd required for blocking transmitter release in response to electrical field stimulation. SCG cultures pre-loaded with [ÅH]-NA released small amounts of radioactivity under resting conditions (L1: 1 00±0 02 %, n = 439 individual cultures) (Fig. 1C) (see also Boehm & Huck, 1995). Electrical field stimulation induced additional release that was prevented by buffer containing either TTX (> 30 nò) (Fig. 1A) or Cd (> 30 ìò) (Fig. 1B). These observations are in line with the conventional concept (Boehm & Huck, 1997) that electrical field stimulation generates Na dependent action potentials (blocked by TTX), which in turn cause Ca entry via VACCs (blocked by Cd, see Boehm & Huck, 1995) and Ca -dependent exocytosis. In agreement with previous reports on chick (Dolezal et al. 1995) and rat sympathetic neurone cultures (Boehm & Huck, 1995), nicotinic agonists induced a concentration-dependent release of [ÅH]-NA that required the presence of Ca in the superfusion buffer (Fig. 1C). However, TTX at concentrations fully competent to block transmitter release by electrical field stimulation (Fig. 1A) was only partially effective when release was evoked by 100 ìò nicotine (Fig. 1D and E) (see also Dolezal et al. 1995). Likewise, Cd at concentrations that entirely blocked either VACCs (Hirning et al. 1988) or electrically induced transmitter release (Fig. 1B) inhibited release in response to nicotine to the same extent as TTX (Fig. 1D). Hence, blockade of voltage-gated Na or Ca channels reveals two components of transmitter release. We call the two components TTX (or Cd ) sensitive and insensitive, respectively.

4 742 D. Kristufek, E. Stocker, S. Boehm and S. Huck J. Physiol In the presence of TTX, transmitter release induced by 100 ìò nicotine was unaffected by 100 ìò Cd (Fig. 1F), indicating a crucial role of voltage-gated Na channels in the signalling cascade that leads from nachr activation to VACC-mediated exocytosis. Hence, under these experimental conditions, depolarization due to nachr activation seems insufficient to gate VACCs directly. The small effect of 300 ìò Cd on TTX-insensitive transmitter release (Fig. 1F) is most probably due to a direct inhibition of nachrs by Cd (Vijayaraghavan et al. 1992; Boehm & Huck, 1995). Nevertheless, we routinely employed this supramaximal Cd concentration for determination of the Cd -insensitive component of transmitter release in order to rigorously rule out any contribution of VACCs. The complementary experiment, testing the effect of TTX on the release induced by 100 ìò nicotine in the presence of 100 ìò Cd, yielded similar results (SµÏS1 ratios of nicotineinduced release in the presence of Cd : 1 03 ± 0 02; SµÏS1 ratios of nicotine-induced release in the presence of Cd, but with 1 ìò TTX added 8 min before and during Sµ: 0 98 ± 0 02; n = 6 individual cultures, P > 0 05, Mann Whitney test). Hence, mechanisms related to the generation of action potentials (such as Na -dependent Ca release from mitochondrial stores, see Rathouz et al. 1996) do not contribute to nicotine-induced transmitter release if VACCs have been blocked by Cd. In sum, these experiments confirm and extend our previous observation that transmitter release in response to nachr activation consists of two components: one that depends on, and one that is independent of VACCs (Boehm & Huck, 1995). TTX and Cd appear to unveil identical aspects of VACC-independent vesicular exocytosis, and we will therefore use the terms TTX- and Cd -insensitive (and TTX- and Cd _sensitive) release in an interchangeable manner. In order to learn more about the properties of the nachrs that form the basis of these two mechanisms we next studied the pharmacology of four prominent nicotinic agonists. Figure 1. [ÅH]-NA release in response to electrical field stimulation or nicotine: effects of TTX and Cd A, effect of TTX on SµÏS1 ratios of transmitter release induced by electrical field stimulation. Indicated concentrations of TTX were added to the superfusion buffer 8 min before and during the second stimulus, Sµ. Means and s.e.m., n = 6 15 individual cultures. B, effect of Cd on SµÏS1 ratios of transmitter release induced by electrical field stimulation. Indicated concentrations of Cd were added to the superfusion buffer 8 min before and during Sµ. Means and s.e.m., n = 6. C, basal transmitter release, and the release induced by 100 ìò nicotine (15 s, indicated by arrows). 0, control buffer containing 2 mò Ca ; 1, superfusion buffer without (arrow at 20 min) or with (arrow at 40 min) 2 mò Ca. Means and s.e.m., n =3.D, effects of 1 ìò TTX or 100 ìò Cd on SµÏS1 ratios of transmitter release induced by 100 ìò nicotine. TTX or Cd was added to the superfusion buffer 8 min before and during Sµ. Means and s.e.m., n = 12 (control, Ctr) or 6 (TTX and Cd ). SµÏS1 ratios in the presence of TTX or Cd do not differ significantly (P > 0 05, Mann Whitney test). E, effect of TTX on SµÏS1 ratios of transmitter release induced by 100 ìò nicotine. Indicated concentrations of TTX were added to the superfusion buffer 8 min before and during Sµ. Means and s.e.m., n =6.F, effect of Cd on SµÏS1 ratios of transmitter release induced by 100 ìò nicotine in the presence of TTX. TTX (1 ìò) was included in the superfusion buffer throughout the collection of the fractions. Indicated concentrations of Cd were added to the superfusion buffer 8 min before and during Sµ. SµÏS1 ratiosinthepresenceof Cd differ from SµÏS1 ratiosofcontrolsonlyat 300 ìò Cd (P < 0 01, Mann Whitney test). Data are means and s.e.m., n =6.

5 J. Physiol Somatic and prejunctional nicotinic receptors 743 Concentration response relationship of transmitter release induced by nicotine, DMPP, cytisine and ACh The concentration response relationship of nicotine-induced transmitter release under control conditions (overall release) and in the presence of 1 ìò TTX (TTX-insensitive release) is shown in Fig. 2. The remarkable relaxation of effects at high agonist concentrations, which results in bell-shaped concentration response curves, is a common phenomenon (see also Fig. 3) that has been noted in several different preparations (see Covernton et al. 1994; Wilkie et al. 1996, and references cited therein). Subtraction of TTX-insensitive release from overall release revealed the TTX-sensitive component (Fig. 2A and B). Curves fitted to the data points of TTX-insensitive release and to calculated TTX-sensitive release revealed EC50 values of 34 6 and 7 6 ìò, respectively (Fig. 2B, Table 1). Hence, the nicotine concentration required to activate the TTX-insensitive component is about fivefold that required to elicit TTX-sensitive transmitter release. In some experiments, the two components of agonistinduced transmitter release were separated by including 300 ìò Cd instead of TTX in the superfusion buffer. The concentration response curve of nicotine-induced transmitter release in the presence of Cd had an EC50 value of 32 8 ± 1 3 ìò and a maximum S% of 3 32 ± 0 12 (estimate ± s.e.m., ALLFIT routine; n = 3 platings), similar to the values obtained for nicotine-induced transmitter release in the presence of TTX (Fig. 2B and Table 1). Hence, the inherent variability between experiments seems to mask the small inhibition (roughly 20 %) of nachrs by 300 ìò Cd mentioned above (Fig. 1F), and we therefore used Cd as an inexpensive substitute for TTX to unveil the TTXinsensitive (now called Cd -insensitive) component. In analogy to the experiments just described for nicotine, release induced by the agonists DMPP, cytisine and ACh in the presence of Cd will produce the Cd -insensitive component, which after subtraction from (overall) release in the absence of Cd discloses the Cd -sensitive component (Fig. 3). The rank order of potency as revealed by these experiments was DMPP > cytisine nicotine ACh for the TTX (or Cd )-sensitive component and DMPP > nicotine > cytisine > ACh for the TTX (Cd )-insensitive Figure 2. Concentration response curves of nicotineinduced TTX-sensitive and TTX-insensitive transmitter release A, nicotine-induced transmitter release under control conditions (overall release, 1) and in the presence of 1 ìò TTX (TTX-insensitive release, 7). Data are means and s.e.m., n = 3 7 platings, each including 3 or 6 individual culture discs. Data points indicated by squares were obtained by subtracting mean values of nicotine-induced TTXinsensitive release (7) from overall release (1) for the same experiment and represent TTX-sensitive release (see text). B, curve fits of TTX-sensitive (±) and TTX-insensitive release (7). Data points were taken from A. Curves were fitted to data points using eqn (1) described in Methods. Apparent affinities for nicotine were 7 60 and 34 6 ìò for TTX-sensitive and TTX-insensitive release, respectively. C, concentration dependence of nicotine-induced transmitter release under control conditions (overall release, as shown in A). The curve was obtained by summation of the two fitted curves for nicotine-induced TTX-sensitive and TTXinsensitive release shown in B.

6 744 D. Kristufek, E. Stocker, S. Boehm and S. Huck J. Physiol Figure 3. Concentration response curves of transmitter release induced by nicotinic agonists Aa, DMPP-induced transmitter release under control conditions (overall release, 1) and in the presence of 300 ìò Cd (Cd -insensitive release, 7). Data are means and s.e.m. (plotted only when exceeding symbol size), n = 4 7 platings, each including 3 individual culture discs, except for 3 and 300 ìò DMPP in the presence of Cd (n = 2). Data points indicated by squares (Cd -sensitive release) were obtained by subtracting DMPP-induced release in the presence of Cd (7) from overall release (1) in the same experiment. The sum of Cd -sensitive and Cd -insensitive release may not exactly match overall release in this figure, since a greater number of experiments were pooled for constructing overall as compared with Cd -sensitive release (note e.g. data points at 100 ìò DMPP). Ab, curve fits of Cd -sensitive (±) and Cd insensitive (7) DMPP-induced transmitter release. Data points were taken from Aa. Curves were fitted to data points using eqn (1). Apparent affinities for DMPP were 3 86 and 22 4 ìò for Cd -sensitiveand Cd insensitive release, respectively. Ba, cytisine-induced transmitter release under control conditions (overall release, 1) and in the presence of 300 ìò Cd (Cd -insensitive release, 7). Data are means and s.e.m., n = 3 platings, each including 3 or 6 individual culture discs. Data points indicated by squares were obtained by subtracting cytisine-induced release in the presence of Cd (7) from overall release (1)forthe

7 J. Physiol Somatic and prejunctional nicotinic receptors 745 component (Tables 1 and 2). These experiments indicate that both components of agonist-induced transmitter release were most potently elicited by DMPP. However, nicotine, cytisine and ACh appeared equipotent in evoking TTX (Cd )-sensitive release, but differed in potency regarding the TTX (Cd )-insensitive component. Due to the dual activation of both nicotinic and muscarinic receptors, effects of ACh are predictably complex. Muscarinic receptor activation inhibits transmitter release (Starke, 1977; Todorov et al. 1991; Boehm & Huck, 1995; Fuder & Muscholl, 1995; Koh & Hille, 1997), VACCs (Bernheim et al. 1992), but also M-type potassium channels (Bernheim et al. 1992) in the rat sympathetic nervous system. Hence, muscarinic receptor activation might explain some specific feature of ACh-induced transmitter release, such as the high EC50 ratio (about 15 instead of 5 as for the other nicotinic agonists) of TTX-insensitive versus TTX-sensitive transmitter release (Fig. 3andTable 1). For a direct evaluation of potency ratios relative to a standard, partial concentration response curves for the four nicotinic agonists were simultaneously evaluated in the same release experiment (low-concentration potency ratios). This approach not only eliminates the variability between different experiments, it also minimizes receptor desensitization due to the lower concentrations of agonist employed. Typical experiments for TTX-sensitive and TTX-insensitive release are shown in Fig. 3D and E, respectively. TTX-sensitive release by this approach was achieved by selecting agonist concentrations previously determined to be too low to cause TTX-insensitive transmitter release (Figs 2 and 3).A summary of these experimentsisprovidedintable 2A (TTX-sensitive release) and C (TTX-insensitive release). Note that potency ratios thus obtained were similar to the potency ratios that were calculated from EC50 values of full concentration response curves (Table 2B and D for TTX-sensitive and TTXinsensitive release, respectively). Data from the experiments used to calculate lowconcentration potency ratios were also analysed by pooling rates of transmitter release at a given concentration of agonist. Hence, the release induced by 1 ìò agonist (TTXsensitive release) was 0 12 ± 0 03 for DMPP, 0 04 ± 0 01 * for cytisine, 0 ** for nicotine, and 0 01 ± ** for ACh (mean S % ± s.e.m.; n = individual cultures; significant difference from DMPP with * P < 0 05 or ** P < 0 01, Mann Whitney test). Release induced by 3 ìò agonist was 1 17 ± 0 21 for DMPP, 0 77 ± 0 09 ** for cytisine, 0 56 ± 0 09 ** for nicotine, and 0 56 ± 0 20 ** for ACh (mean S % ± s.e.m.; n = 18 20; difference from DMPP significantwith**p< 0 01, Mann Whitney test). Rates for TTX-insensitive transmitter release (i.e. in the presence of 1 ìò TTX, see Fig. 3E) in response to 15 ìò agonist were 0 35 ± 0 05 for DMPP, 0 08 ± 0 01 ** for cytisine and 0 12 ± 0 02 ** for nicotine (mean S % ± s.e.m.; n = 11 12; difference from DMPP significant with ** P < 0 01, Mann Whitney test). Hence, at a given concentration of the agonist, DMPP evoked significantly more TTX-sensitive as well as TTXinsensitive release than any of the other agonists, including cytisine. These data, as well as the calculated potency ratios (Fig. 3D and E and Table 2), translate into DMPP being the most potent of the agonists for both TTX-sensitive and same experiment. Bb, curve fits of Cd -sensitive (±) and Cd -insensitive (7) cytisine-induced transmitter release. Data points were taken from Ba. Curves were fitted to data points using eqn (1). Apparent affinities for cytisine were 7 01 and 48 7 ìò for Cd -sensitive and Cd -insensitive release, respectively. Ca,ACh-induced transmitter release under control conditions (overall release, 1) and in the presence of 300 ìò Cd (Cd -insensitive release, 7). Data are means and s.e.m., n = 3 8 platings, each including 3 individual culture discs. Data points indicated by squares were obtained by subtracting ACh-induced release in the presence of Cd (7) from overall release (1) forthesameexperiment.cb, curve fits of Cd sensitive (±) and Cd -insensitive (7) ACh-induced transmitter release. Data points were taken from Ca. Curves were fitted to data points using eqn (1). Apparent affinities for ACh were 7 45 and ìò for Cd -sensitive and Cd -insensitive release, respectively. D, partial concentration response curves of TTXsensitive transmitter release induced by DMPP (9), cytisine (7), nicotine (1) and ACh(±)in a single experiment made up of 12 individual culture discs. Indicated concentrations of agonists, each tested in one triplet of culture discs, were added at 20 min intervals. Data points are means from 3 individual discs. Error bars (s.e.m.) did not exceed symbols. Curves were simultaneously fitted to data points with the ALLFIT routine with the constraints of a shared slope and a fixed maximum as described in Methods. Potency ratios relative to the standard (DMPP) were 0 51 for nicotine, 0 47 for cytisine and 0 36 for ACh. Averaged potency ratios from identically designed experiments are provided in Table 2A. Note that agonist concentrations employed will only induce TTX-sensitive transmitter release. E, partial concentration response curves of TTX-insensitive transmitter release induced by DMPP (9), cytisine (7), nicotine (1) and ACh(±) in a single experiment in the presence of 1 ìò TTX. Agonists at indicated concentrations were each added to one triplet of culture discs as described for D. Data points are means from 3 individual culture discs. Error bars (s.e.m.) did not exceed symbol size. Curves were simultaneously fitted to data points with the ALLFIT routine with the constraints of a shared slope and a fixed maximum. Potency ratios relative to the standard (DMPP) were 0 87 for nicotine, 0 61 for cytisine and 0 14 for ACh. Averaged potency ratios from identically designed experiments are provided in Table 2C.

8 746 D. Kristufek, E. Stocker, S. Boehm and S. Huck J. Physiol Table 1. Effects of saturating concentrations of nicotinic agonists on induction of transmitter release and whole-cell currents DMPP (27) Cytisine (22) Nicotine (14) ACh (10) EC50 p Emax EC50 p Emax EC50 p Emax EC50 p Emax (ìm) (ìm) (ìm) (ìm) A. Current ** 1 41 ** * ** ** 1 52 ** ** ± 0 7 ± 0 06 ± 5 2 ± 1 8 ± 0 03 ± 6 7 ± 1 7 ± 0 07 ± 8 3 ± 6 7 ± 0 07 ± 11 4 B. TTX-sensitive release ± 1 94 ± 1 57 ± 1 88 ± 1 66 ± 0 46 ± 0 30 ± 0 44 ± 0 42 ± 0 01 ± 1 94 ± 3 40 ± 0 06 C. TTX-insensitive release ± 4 6 ± 2 07 ± 0 20 ± 9 22 ± 0 78 ± 0 32 ± 1 2 ± 1 01 ± 0 13 ± 14 1 ± 0 27 ± 0 14 D. Ratio insensitiveï sensitive A, EC50 values, slope factors (p) and maximal effects (Emax, papf ) for agonist-induced currents recorded by the perforated patch technique as described in Methods. Representative original current traces with concentration response curves are shown in Fig. 4. Data points are the averaged parameters (± s.e.m.) from curvefitsthatwereconstructedforeachindividualcell(number of cells is given beside the agonist in parentheses). Statistical differences relative to the effects of DMPP were calculated by the Mann Whitney test (* P < 0 05; ** P < 0 01). From this summary it appears that maximal effects of both nicotine and DMPP were smaller than those of ACh and cytisine. DMPP effects were also consistently smaller than either cytisine- or ACh-induced peak currents when compared in identical cells (cytisine, 106 ± 10 pa pf ; DMPP, 74±6pApF ; means ± s.e.m.; P < 0 01, Wilcoxon test, n = 17; ACh, 116 ± 11 pa pf ; DMPP, 69±9pApF ; P < 0 01, Wilcoxon test, n = 10; see also Fig. 4Aa and Ab). By contrast, a direct comparison of maximal effects of nicotine- and cytisine-induced currents in identical cells revealed no significant difference between the two substances (nicotine, 77±5pApF ; cytisine, 78±5pApF ; means ± s.e.m.; P > 0 05, Wilcoxon test, n = 5; see also Fig. 4Ba and Bb). Averaged cell capacitance was 33 ± 1 pf (mean ± s.e.m.), n = 41 cells. B, data points taken from curve fits of Figs 2and 3 (Emax, S %). TTX-sensitive release also stands for Cd -sensitive release. C, data points taken from curve fits of Figs 2 and 3. TTX-insensitive release also stands for Cd -insensitive release. D, ratio of TTX-insensitiveÏTTXsensitive transmitter release. Note that except for ACh, the ratio of EC50 values varies by a factor of about 6 (range, ). TTX-insensitive transmitter release. Since this rank order of potency clearly differs from previous observations made by patch clamp recordings in (adult) rat SCG ganglia (Covernton et al. 1994), we recorded agonist-induced currents in dissociated ganglia of postnatal rats to complement our release experiments. Whole-cell currents induced by DMPP, nicotine, cytisine and ACh Our transmitter release experiments employed a wide range of agonist concentrations sufficient to reach a maximum response, but also partial dose response curves that directly related the potencies of three agonists to a standard, DMPP. In order to match these observations with patch clamp experiments, we established both full and partial concentration response curves of agonist-induced whole-cell currents. A comparison of EC50 values (inferred from full-range dose response curves) revealed DMPP as the most and ACh as the least potent substance for inducing currents (Fig. 4 and Tables 1 and 2), which parallels effects on transmitter release. However, DMPP was also the least efficacious of the four agonists when tested in patch clamp recordings (see legend to Table 1). A reduced maximal effect of DMPP compared with ACh has been noted previously (Mandelzys et al. 1995) and may be caused by both a pronounced channel block and receptor desensitization (Mathie et al. 1991) at high concentrations of DMPP. As a result, such high agonist concentrations can produce misleading concentration response curves (Covernton et al. 1994), and we therefore extended our experiments to make comparisons of relative potencies at non-saturating concentrations of agonists. In fact, rank orders changed significantly when recording whole-cell currents in response to low concentrations of nicotinic agonists. In line with a previous report (Covernton et al. 1994), cytisine now appeared to be the most potent agonist, followed by DMPP nicotine, and ACh (Fig. 5and Table 2). In order to make our data comparable with previous observations (Covernton et al. 1994; Mandelzys et al. 1995) we started with conventional measurements of agonistinduced peak currents. However, transmitter release in response to nicotinic agonists might be better accounted for by choosing the charge transfer (measured by the time

9 J. Physiol Somatic and prejunctional nicotinic receptors 747 Table 2. Potency ratios of cytisine, nicotine and ACh relative to DMPP for agonist-induced transmitter release (A D) and agonist-induced currents (E G) DMPP Cytisine Nicotine ACh A. TTX-sensitive release, partial dose response ± 0 03 (7) * 0 56 ± 0 08 (6) * 0 39 ± 0 05 (7) * B. TTX-sensitive release, full dose response C. TTX-insensitive release, partial dose response ± 0 06 (4) ** 0 79 ± 0 08 (4) n.s ± 0 05 (4) ** D. TTX-insensitive release, full dose response E. Peak current, partial dose response ± 0 07 (17) ** 0 92 ± 0 04 (17) n.s ± 0 03 (17) ** F. Time integral of current, partial dose response ± 0 07 (17) ** 0 98 ± 0 04 (17) ** 0 56 ± 0 03 (17) ** G. Peak current, full dose response A, potency ratios of agonist-induced, TTX-sensitive transmitter release using DMPP as a standard. The 4 nicotinic agonists were simultaneously tested in the same experiment at concentrations (3 and 5 ìò) that will only produce submaximal TTX-sensitive release (see Figs 2 and 3). Potency ratios for individual experiments were calculated from curves simultaneously fitted to data points with logistic equations that were constrained to be parallel and to fixed maxima implied from full concentration response curves (see Methods). Data are means ± s.e.m. from 6 7 release experiments with 3 individual culture discs for each substance (one of these experiments is shown in Fig. 3D). * Significantly different from 1 (P < 0 05, Wilcoxon test). Potency ratios of cytisine and ACh (P < 0 05), but not of nicotine and ACh (P > 0 05, Wilcoxon test) differ significantly. Note that numbers < 1 mean potencies less than DMPP. B, potency ratios calculated from EC50 values of TTX-sensitive transmitter release shown in Figs 2and3and Table 1. C, potency ratios for TTX-insensitive transmitter release (i.e. in the presence of 1 ìò TTX). The 4 nicotinic agonists were simultaneously tested in the same experiment at concentrations that will only produce submaximal TTX-insensitive release (see Figs 2 and 3). Potency ratios for individual experiments were calculated from curves simultaneously fitted to data points with logistic equations that were constrained to be parallel and to fixed maxima implied from full concentration response curves (see Methods). Data are means ± s.e.m. from 4 release experiments with 3 individual culture discs for each substance (one of these experiments is shown in Fig. 3E). ** Significantly different from 1 (P < 0 01); n.s., not significantly different from 1 (P > 0 05), Wilcoxon test. D, potency ratios calculated from EC50 values of TTX-insensitive transmitter release shown in Figs 2 and 3 and Table 1. E, potency ratios deduced from agonist-induced peak currents. Cells were exposed to the 4 nicotinic agonists at the low-concentration end of the dose response curve. Potency ratios were calculated from the peak currents elicited in individual cells (see Fig. 5B for an example) by curves simultaneously fitted to data points. Curve fits were performed with logistic equations that were constrained to be parallel and to a fixed maximum implied from full concentration response curves (see Methods). Data are means ± s.e.m. of the ratios of 17 individual cells from 5 different platings. ** Significantly different from 1 (P < 0 01); n.s, not significantly different from 1 (P > 0 05), Wilcoxon test. F, potency ratios deduced from 15 s time integrals of agonist-induced currents. Data points are from the same experiments described in E except that 15 s time integrals instead of maxima of currents were used for the calculation of potency ratios. ** Significantly different (P < 0 01) from the respective potency ratio shown in E (Wilcoxon test). G, potency ratios calculated from the EC50 values (provided in Table 1) of full concentration response curves of agonist-induced currents. integral of currents) that occurs during the application of an agonist. Slopes of full-range dose response curves (as shown in Fig. 4) thus determined became significantly steeper for all agonists tested (P < 0 01, Wilcoxon test), and EC50 values were lower particularly for DMPP (from 20 1 to 14 8 ìò; P < 0 01, Wilcoxon test), but also for ACh (from 66 4 to 62 5 ìò, P < 0 05, Wilcoxon test). Steeper slope factors and a leftward shift of dose response curves were to be expected, since the time integral of currents (even for drug applications as short as 1 s) will more sensitively reflect a desensitization phenomenon than measurements of peak current. Likewise, potency ratios determined in response to agonists at the low-concentration end of the dose response curve were significantly affected when taking the time integral instead of the maxima of currents (Fig. 5 and Table 2). For example, cytisine was now more potent by a factor of 1 61 (instead of 1 44, P < 0 01, Wilcoxon test) than DMPP

10 748 D. Kristufek, E. Stocker, S. Boehm and S. Huck J. Physiol (Table 2F). Since time integrals of currents were taken from a 15 s period of drug application, these observations best match our transmitter release experiments that also used 15 s exposure times of agonists. In sum, nicotinic agonists followed a rank order of potency of cytisine>nicotine DMPP>ACh when tested with patch clamp recordings at the low-concentration end of the dose response curve. A different rank order of potency (DMPP nicotine > cytisine > ACh) resulted when exploring the full range of dose response curves. However, due to pronounced desensitization at high agonist concentrations that will distort dose response curves, potency ratios deduced from such experiments may be misleading (Covernton et al. 1994). Effects of the nicotinic antagonists mecamylamine, DHâE and á_butx The nicotinic antagonist mecamylamine (Ascher et al. 1979; Lindstrom et al. 1996) inhibited overall release (induced by 100ìÒnicotine)withanIC50 value of ìò, and Cd insensitive release (induced by 100 ìò nicotine in the presence of 300 ìò Cd ) with an IC50 value of ìò (Fig. 6). Full inhibition of overall and Cd -insensitive transmitter release was observed with 10 and 1 ìò mecamylamine, respectively (Fig. 6C), indicating that release in response to nicotine is specifically coupled to the activation of nachrs. The effects of mecamylamine on overall release were clearly dependent on concentrations of both the agonist and the antagonist (Fig. 6A). Hence, 100 nò mecamylamine inhibited the release in response to 30 ìò nicotine by only 11 %, whereas release induced by 100 ìò nicotine was inhibited by about 60 % (Fig. 6A and C). These observations are compatible with the concept of an open-channel, usedependent block (Ascher et al. 1979; Gurney & Rang, 1984). With Cd -insensitive transmitter release, the effects of 1 ìò mecamylamine were insurmountable even by excessive concentrations of nicotine (Fig. 6B), which indicates a noncompetitive mode of action. An incomplete, non-competitive Figure 4. Examples of agonist-induced whole-cell currents at a concentration range that covers saturating responses Aa and Ba, original traces of currents induced by DMPP, ACh, nicotine and cytisine in 2 different cells (A and B). Pairs of indicated agonists were tested in cells voltage clamped at 70 mv using the perforated patch technique as described in Methods. Agonists were applied at concentrations indicated in Ab and Bb. Ab and Bb, dose response relationship of peak currents constructed from the original recordings shown in Aa and Ba. Data points are mean peak currents measured in duplicate (±, ACh; 9, DMPP; 1, nicotine; 7, cytisine). Curves fitted to data points by means of eqn (1) revealed EC50 values of 19 7 ìò for DMPP, 96 4 ìò for ACh, 28 5 ìò for nicotine and 22 2 ìò for cytisine. A summary of related experiments is provided in Table 1A.

11 J. Physiol Somatic and prejunctional nicotinic receptors 749 inhibition of Cd -insensitive transmitter release was seen in the presence of 30 nò mecamylamine (Fig. 6B). Mecamylamine at 1 ìò did not affect the release induced by electrical field stimulation (control SµÏS1 ratio: 0 91 ± 0 02; SµÏS1 ratio with 1 ìò mecamylamine added 8 min before and during Sµ: 0 90 ± 0 03, n = 3 individual cultures). Given their exceptional Ca permeability, á_butx-sensitive nachrs composed of á7 subunits (McGehee & Role, 1995b) would be particularly suitable to mediate TTX-insensitive transmitter release. However, we found 50 nò á_butx to be without effect on the TTX-insensitive component (control SµÏS1 ratioswith100ìònicotineinthepresenceof1ìò TTX: 0 77 ± 0 01; SµÏS1 ratios with á_butx added 16 min before and during Sµ: 0 81 ± 0 02; n = 15; P > 0 05, Mann Whitney test). It has previously been shown in rat SCG neurones that DMPP-induced currents and the associated increase of intracellular Ca are unaffected by á_butx (Trouslard et al. 1993). Competitive antagonists are valuable tools for the characterization of receptors (Arunlakshana & Schild, 1959), and one of the best-described competitive antagonists of the nachr is DHâE (Harvey et al. 1996). Initial experiments indicated a rightward shift without noticeable depression of the dose response curve of nicotine-induced, overall transmitter release in the presence of 50 ìò DHâE (not shown). We wanted to know whether DHâE might differentially affect TTX-sensitive and TTX-insensitive transmitter release and therefore investigated effects of the Figure 5. Examples of agonist-induced whole-cell currents elicited at the low-concentration end of the dose response curve A, original traces of currents induced by DMPP, ACh, nicotine and cytisine. Currents were recorded in the same cell by the perforated patch technique as described in Methods, except that application times were extended to 15 s. The cell was voltage clamped at 70 mv. Apart from ACh (4 ìò), agonist concentrations for the effects shown were 3 ìò. B, dose response relationship of peak currents constructed from original recordings as shown in A. Data points are mean peak currents measured in duplicate for each agonist. Curves based on eqn (1) were simultaneously fitted to data points by weighted non-linear regression using the ALLFIT routine with the constraints of a shared slope and a fixed maximum as described in Methods. Potency ratios relative to the standard (DMPP, 9) were 1 33 for cytisine (7), 0 69 for nicotine (1) and 0 46 for ACh (±). Note that numbers > 1 mean potencies greater than DMPP (i.e. larger effects at equal concentrations). Averaged potency ratios from identically designed experiments are provided in Table 2E. C, dose response relationship constructed from original recordings as shown in A, but based on a 15 s time integral (i.e. the area under the curve during a 15 s application of agonist) instead of measuring peak currents as shown in B. Data points are mean 15 s time integrals of currents measured in duplicate for each agonist. Potency ratios relative to DMPP (9) were 1 61 for cytisine (7), 0 84 for nicotine (1) and 0 57 for ACh (±), calculated by the same method as described in B. Averaged potency ratios from identically designed experiments are provided in Table 2F.

12 750 D. Kristufek, E. Stocker, S. Boehm and S. Huck J. Physiol substance by protocols that would separate the two components of transmitter release (Fig. 7). Schild plots constructed from curve shifts (dose ratios) of nicotine dose response curves in the presence and absence of DHâE (Fig. 7A and C) revealed paµ values of 4 03 and 4 25 for TTX-sensitive and TTX-insensitive transmitter release, respectively (Fig. 7B and D). For TTX-sensitive release, the slope of the Schild regression did not significantly differ from unity ( 0 87, Fig. 7B), whereas the regression line appeared significantly steeper than the expected value of 1 for TTX-insensitive release ( 1 13, Fig. 7D). Possible explanations for this deviation from unity might be antagonist concentrations too low for an accurate estimate, or non-equilibrium conditions of the antagonist applied in these experiments (Fig. 7A and D). In any case, the rather close paµ values of 4 03 and 4 25 indicate that DHâE does not seem to discriminate between nicotinic receptors that trigger transmitter release by a TTX-sensitive versus a TTX-insensitive mechanism. We argued above that due to differences in the rank order of agonists somatic nachrs differ from release-mediating prejunctional receptors. It was therefore of interest to compare the effects of DHâE on nicotine-induced currents with its effects on nicotine-induced transmitter release. In line with the known action of DHâE as a competitive antagonist (Harvey et al. 1996), the substance caused a rightward shift without noticeable depression of the dose response curve of nicotine-induced currents (Fig. 7E). The paµ value of 4 58 calculated from the Schild plot of these experiments (Fig. 7F) is somewhat higher than the values obtained for TTX-sensitive (4 03, Fig. 7B) and TTXinsensitive (4 25, Fig. 7D) transmitter release. However, considering much larger differences when testing DHâE on recombinant nachrs in heterologous expression systems (Harvey et al. 1996), this difference appears too small to make a distinction between somatic and prejunctional receptors by the use of this antagonist. Figure 6. Effects of mecamylamine on nicotine-induced transmitter release A, nicotine-induced transmitter release under control conditions (overall release, 1), and in the presence of 100 nò mecamylamine (0), 300 nò mecamylamine (8)and1ìÒmecamylamine(6). Note that inhibition by mecamylamine depends on the concentration of nicotine. Data are means and s.e.m. (only shown when exceeding symbol size), n = 6 individual cultures. B, insurmountable inhibition of Cd -insensitive transmitter release by 30 nò and 1 ìò mecamylamine. Transmitter release was induced by the indicated concentrations of nicotine in the presence of 300 ìò Cd and in the absence (control, 1)orcontinuouspresenceof30nÒ(0) or 1 ìò(6) mecamylamine. Data are means ± s.e.m. (only shown when exceeding symbol size), n = 3 5. C, effects of mecamylamine on overall and on Cd -insensitive, nicotine-induced transmitter release. Transmitter release was induced by 2 successive stimuli of 100 ìò nicotine (S1, Sµ) in the absence (overall release, 9)or presence of 300 ìò Cd (Cd -insensitive release, 7) and indicated concentrations of mecamylamine added to the superfusion buffer 8 min before and during Sµ. SµÏS1 ratiosinthepresenceof mecamylamine were compared with SµÏS1 ratios under control conditions. Data are mean values of percentage inhibition (s.e.m. did not exceed symbol size), n = 3 6. The curves were fitted to data points by means of eqn (1) with fixed parameters for maximum (100 % inhibition) and minimum (0 %). Curve fits yielded IC50 values of and ìò mecamylamine for overall and Cd -insensitive release, respectively.