Journal of Physiology (1991), 437, pp. 201-220 With 9 figures Printed in Great Britain

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DISSOCIATION BETWEEN INTRACELLULAR Ca2+ AND MODULATION OF [3HJNORADRENALINE RELEASE IN CHICK SYMPATHETIC NEURONS

By DENNIS A. PRZYWARA, SANJIV V. BHAVE, ANJALI BHAVE, TARUNA D. WAKADE AND ARUN R. WAKADE From the Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, MI 48201, USA

(Received 18 May 1990) SUMMARY

1. We studied the relation between cyclic AMP, intracellular Ca2" concentration and release of [3H]noradrenaline ([3H]NA) in sympathetic neurons cultured from chick embryos. 2. Forskolin (10 ,tM) and vasoactive intestinal polypeptide (VIP, 3/M) increased cellular levels of cyclic AMP 8- and 3-fold, respectively, either in the absence or presence of electrical stimulation. Electrical stimulation (1 Hz for 10 s) alone had no effect on cyclic AMP levels. 3. Electrically evoked (1 Hz for 10 s) release of [3H]NA was facilitated by 10 ,tMforskolin, 3 1um-VIP and by the non-hydrolysable cyclic AMP analogue, 8bromoadenosine 3': 5'-cyclic monophosphate (8-Br-cyclic AMP). The inactive analogue of forskolin, dideoxyforskoli-n, had no effect on [3H]NA release. 4. The stimulation-evoked release of [3H]NA was completely inhibited by the neuronal blocking drugs guanethidine (1 /M) and bretylium (3 /tm). 5. Whole-cell voltage-clamp studies showed that forskolin and VIP did not facilitate and guanethidine and bretylium did not block voltage-activated Ca2" currents in the cell bodies of sympathetic neurons. 6. Fluorescence measurements using the Ca2"-sensitive dye Indo-1 revealed that forskolin and guanethidine had no effect on the electrically stimulated increase in intracellular Ca2+ concentration recorded from the cell bodies and the growth cones. 7. We conclude that release of [3H]NA can be enhanced or blocked without affecting the increase in intracellular Ca2+ concentration produced by electrical stimulation. Therefore, it is possible that pharmacological agents enhance or depress the release of [3H]NA by acting on steps of exocytosis that are down-stream from Ca2` mobilization. INTRODUCTION

Modulation of transmitter release has long been believed to be a direct consequence of changes in [Ca2+]i in secretory cells (Douglas, 1968; Katz, 1969; Kirpekar, 1975). Agents which block the release of [3H]noradrenaline ([3H]NA) from sympathetic neurons are thought to do so, either directly or indirectly, by blocking the stimulated MS 8507

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rise in [Ca2+]i. Agents which directly block neuronal Ca2" channels are, in general, effective blockers of neurotransmitter release. The neuronal blocking agents guanethidine and bretylium have not been shown to block Ca2" channels but are believed to exert a selective local anaesthetic action on sympathetic neurons and thus inhibit the depolarization-induced Ca2+ influx and transmitter release (Mitchell & Oates, 1970). Conversely, the effects of Ca2+ channel blocking drugs which do not block transmitter release (dihydropyridines) are used to support the hypothesis that NA release is coupled specifically to dihydropyridine-resistant (N-type) Ca2+ channels in sympathetic neurons (Hirning, Fox, McCleskey, Olivera, Thayer, Miller & Tsien, 1988). Agents which facilitate neurotransmitter release have also been proposed to act by increasing [Ca2+]i in sympathetic neurons. Forskolin is a well-known activator of adenylate cyclase (Seamon, Padgett & Daly, 1981) and also facilitates the release of [3H]NA from sympathetic neuroeffector organs (Cubeddu, Barnes & Weiner, 1975;* Stjarne, 1976; Gothert & Hentrich, 1984; Wakade, Malhotra, Wakade & Dixon, 1986). In cardiac cells it is well documented that activation of adenylate cyclase and increased cyclic AMP levels result in increased Ca2+ influx via voltage-sensitive Ca2+ channels (Osterrieder, Brum, Hescheler, Trautwein, Flockerzi & Hofmann, 1982; Reuter, 1983). This concept has been extrapolated to explain the facilitatory effects of forskolin on the release of [3H]NA from neuroeffector organs. However, presynaptic Ca21 influx could not be measured in these preparations. Recent work with invertebrate neurons has shown that transmitter release can be modulated independently of changes in [Ca2+]i (Man-Son-Hing, Zoran, Lukowiak & Haydon, 1989; Dale & Kandel, 1990). One mechanism by which neurotransmitter release can be modulated down-stream from Ca2+ entry is phosphorylationdependent regulation of proteins associated with synaptic vesicles. Synapsin I is a major nerve terminal phosphoprotein which is specifically associated with synaptic vesicles (DeCamilli, Harris, Huttner & Greengard, 1983). It is a substrate for both cyclic AMP-dependent and type II Ca2+-calmodulin-dependent kinase (Nairni Hemmings & Greengard, 1985; DeCamilli & Greengard, 1986). In the squid giant synapse it has been shown that microinjection of dephosphorylated synapsin I or calmodulin kinase II decreased or increased, respectively, transmitter release with no effect on the voltage-dependent Ca21 current (Llinas, McGuinness, Leonard, Sugimori & Greengard, 1985). Ca2+-independent modulation of secretion has also been reported to occur, via GTP-binding proteins, in permeabilized secretory cells (Barrowman, Cockcroft & Gomperts, 1986; Luini & DeMatteis, 1990). Ca2+independent modulation of transmitter release has not been studied in intact vertebrate sympathetic neurones. We have previously shown that [3H]NA release from sympathetic neurons cultured from embryonic chick is dependent on the presence of extracellular Ca2+ (Wakade & Wakade, 1982, 1988). In the present work we take advantage of Ca2+imaging techniques and whole-cell voltage clamp to monitor Ca2+ influx and [Ca2+]i in pure cultures of sympathetic neurons under conditions which enhance (forskolin, VIP and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP)) and block (guanethidine and bretylium) transmitter release. These studies provide direct evidence that release of [3H]NA can be enhanced or blocked without affecting the rise

Ca20-IND)EPENDENT MODULATION OF EXOCYTOSIS 203 in [Ca2+]i during stimulation. Our results suggest that the role of cyclic AMP in enhancing the release of [3H]NA is in the utilization rather than the mobilization of Ca2" ions by sympathetic neurons. Some of the results were presented to the 11th International Congress of Pharmacology held in Amsterdam (Wakade, Wakade, Bhave, Bhave, Shukla & Przywara, 1990b). METHODS

[3HJNA release Sympathetic neurons derived from sympathetic ganglia of 10-day-old chick embryo (75000 cells/dish) were used after 2 days in culture. Culture conditions yielding pure populations of sympathetic neurons were as previously described by Wakade, Edgar & Theonen (1982) with the modifications of Wakade, Bhave, Malhotra & Wakade (1990a). Neurons were loaded with [3H]NA (3 ,uCi/ml Krebs solution, 43-9 Ci/mmol; New England Nuclear, Boston, MA, USA) for 60 min in a CO2 incubator. After 60 min wash-out, samples were collected, over 2 min periods, in the control medium to establish spontaneous release of [3H]NA. Neurons were stimulated at 1 Hz for 10 s (120 mA, 1-0 ms duration applied via platinum-wire electrodes connected to a Grass stimulator) to determine evoked [3H]NA release. Samples were then collected before and after stimulation in the presence of the indicated test agent. Each agent was in contact with neurons for 10 min before collection of samples unless otherwise noted. For details of studies on [3H]NA release from cultured neurons see Wakade & Wakade, 1988.

Cyclic AMP and protein determination The cyclic AMP content of neurons treated as reported in the Results section was determined as previously described (Wakade, Wakade, Bhave & Malhotra, 1988). Briefly, cells were extracted, homogenized and centrifuged in ice-cold, 10 % trichloracetic acid (TCA). The clear supernatant was treated with water-saturated ether to remove TCA. The extract was evaporated to dryness, reconstituted in sodium acetate buffer and assayed for cyclic AMP content using a radioimmunoassay kit (New England Nuclear, Boston, MA, USA). For protein determination the TCA extract was centrifuged and the precipitate was resuspended in 1 M-NaOH and assayed according to the methods of Schacterle & Pollack (1973). Ca2, currents Peak voltage-elicited Ca2+ current (Ica) was recorded from the same neuron during control and the indicated drug treatment. Cover-slips with attached sympathetic neurons were placed in a 15 ml superfusion chamber mounted on the stage of an inverted microscope (Nikon diaphot with Hoffman Contrast Modulation optics). The bathing solution contained (in mM): 120 NaCl, 4-7 KCl, 1 MgCl2, 5 CaCl2, 10 HEPES buffer, 10 glucose, 20 tetraethylammonium-Cl (TEA; Eastman Kodak, Rochester, NY, USA), and 200 nM-tetrodotoxin (TTX; Calbiochem-Behring, La Jolla, CA, USA), pH 7 3 with NaOH. Electrodes of 0-8 to 3 MQ resistance contained (in mM): 100 CsCl, 5 MgCl2, 10 EGTA, 2 Mg-ATP, 40 HEPES buffer, pH 7-3 with CsOH. Cells were voltage clamped at room temperature (22-25 °C) using the whole-cell variation of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Cells were tested for current run-down before exposure to test agents and only those showing stable currents were used. Drugs were added by superfusing 5 ml of desired solution through the bath. Inward currents were elicited when cells held at -70 mV were depolarized to 0 mV for 200 ms. Data for current-voltage curves were obtained .when cells held at -70 mV were depolarized, in 10 mV steps, to + 60 mV. Twenty seconds was allowed between sequential step depolarizations. The signal from the patch-clamp amplifier (EPC7, List-electronic, Germany) was filtered at 3 kHz through an 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA) and digitally stored and analysed using a personal computer (TL1-125 kHz DMA interface and pCLAMP software, Axon instruments, Foster City, CA, USA). Neurons with extensive processes cannot be voltage clamped. However, cells without neurites are not representative of functional neurons. For these reasons neurons which possessed a single neurite were selected for the voltage-clamp experiments. Most of these cells appeared to be adequately voltage clamped. That is, currents showed a smooth onset without notches and no longlasting tails upon repolarization. Cells exhibiting signs of inadequate space clamp were not used.

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To ensure the accuracy of voltage-clamp data recorded at the neuronal cell body, experiments were performed to determine the effects of unclamped current flow along the neuronal process. ICa activation was recorded at membrane potentials between -30 and + 1O mV from a holding potential of -70 mV. A pulled glass capillary pipette, polished closed at the tip (approximate tip diameter 5,um) was then used to gently crush the neuronal process and block conduction at a distance along the process approximately equal to the diameter of the cell body. ICa activation was tested again 10 min after the crush-induced conduction block. Intracellular free Ca2+ concentration [Ca2+]i was measured at rest and during electrical stimulation in control and drug containing solution. Cell bodies and growth cones were selected to show that each area is capable of exhibiting a Ca2+ response to electrical stimulation. Sympathetic neurons cultured on the glass cover-slips were loade'l with 0 25 /tM-Indo- 1-acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) for 60 min anid washed 2 times with Krebs solution at 37 'C. From this point on the cells were maintained in HEPES solution at room temperature. The cover-slip was placed in a Leiden chamber and secured on the stage of an ACAS laser photometer (Meridian Instruments, Lansing, MI, USA). Neurons were illuminated by laser light of 340-360 nm and Indo- fluorescence recorded at 405 nm (Ca2+-bound) and 485 nm (Ca2`-free) wavelengths. The fluorescence ratio (450/485 nm) was used to eliminate possible artifacts due to variations in thickness or Indo-1 distribution in the cells (Grynkiewiez, Poenie & Tsien, 1985). Neutral density filtering reduced photobleaching and allowed reproducible responses from the same sites. [Ca2+]i was calculated by computer from a standard curve of fluorescence ratio versus free Ca2+ in Ca2+-EGTA buffer. Recording and optical parameters were identical for cell bodies and growth cones except for scan strength of 10-20 % and 40-60% for bodies and growth cones, respectively. Three protocols were used to monitor [Ca2+]i, image analysis, point analysis and line average. Image analysis. This mode was used to monitor Ca2+ distribution in the entire cell body or growth cone under investigation. Simultaneous images at the two wavelengths were obtained over 10 s scanning periods at rest and coincident with the 10 s electrical stimulation, identical to the protocol used for evoked [3H]NA release. Images from cell bodies and growth cones were recorded at a step size of 0-75 and 0-25,um, respectively. Decrease in the step size (the distance between two scanning points along the x- and y-axis) provides an enlarged image at the same optical setting. All images shown in Results are at 400 x magnification. Point analysis. The point analysis protocol was used to monitor dynamic changes in [Ca2+]i on a milliseconds time scale. A point (3-6 1am2) was selected in the cytosolic region of cell bodies or growth cones. Data were collected simultaneously from both detectors at 5 ms intervals during the scanning period and converted to Ca2+ concentrationr. Various cytosolic locations from near the cell membrane to the mid-central region of the cell body or growth cone were monitored. Line average. This protocol provided Ca2+ distribution data at a time resolution intermediate between that of whole image and point analysis. The co-ordinates of the scan line (Xl, Yl to X2, Y1) were chosen such that the scan approximated the diameter of the cell body but did not pass through the nucleus. The width of the line was approximately 3-5 ,um. The left-to-right scan was complete in 10 ms and repeated at 500 ms intervals. The traces of [Ca2+]i versus time presented in results show the average [Ca2+]i recorded during each 10 ms scan across the same line, i.e. a[Ca2+]i value was obtained every 500 ms throughout the scanning period. [Ca2+]i was monitored in neurons at rest (20s), during 10 s electrical stimulation and during recovery (20 s). The protocol was then repeated during exposure to the indicated drugs. 1

Drugs The following drugs were used: forskolin (Calbiochem, San Diego, CA, USA) dissolved in dimethyl sulphoxide; vasoactive intestinal polypeptide (VIP), 8-bromoadenosine 3': 5'-cyclic monophosphate (8-Br-cyclic AMP) and tetracaine hydrochloride (Sigma, St Louis, MO, USA); guanethidine (Ciba-Geigy, Summit, NJ, USA); bretylium tosylate (Burroughs Wellcome, Research Triangle Park, NC, USA).

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RESULTS

Cyclic AMP content of sympathetic neurons Although cyclic AMP has been implicated in the modulation of NA release from sympathetic neurons (Starke, 1987), no direct evidence is available. Our first goal was to measure cyclic AMP content in resting and stimulated sympathetic neurons. As shown in Fig. 1, cyclic AMP levels were not changed during field stimulation (1 Hz for 10 s). This figure also shows that the well-known activator of adenylate cyclase, ._C

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forskolin (10 gM), produced an 8-fold increase in neuronal cyclic AMP. It has been reported that cyclic AMP levels are elevated by activation of membrane receptors for VIP (Volle & Patterson, 1982; Malhotra, Wakade & Wakade, 1989). In the present study, VIP (3 /M) produced a 3-fold increase in cyclic AMP levels (Fig. 1). Although not shown, higher concentrations of VIP (10 /M) further increased the cyclic AMP content by about 5-fold compared to control.

Facilitation of [3H]NA release by cyclic AMP enhancers When tested under the same experimental conditions that lead to enhanced cyclic AMP levels forskolin and VIP markedly facilitated electrically stimulated release of tritium from [3H]NA-loaded sympathetic neurons (Fig. 2). The cyclic AMP analogue, 8-Br-cyclic AMP (1 mM) also produced a significant facilitation of [3H]NA release during stimulation (Fig. 2). The inactive forskolin analogue, dideoxyforskolin, was used to further implicate the involvement of cyclic AMP in facilitation of [3H]NA release. This agent had no effect on the evoked release (not shown). None of the above test agents enhanced the release of [3H]NA during the non-stimulation period.

D. A. PRZYWARA AND OTHERS

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8-Br-cAMP Control Forskolin VIP Fig. 2. Agents which enhance or mimic cyclic AMP also enhance the electrically evoked release of [3H]NA from sympathetic neurons. Fractional release of [31H]NA was determined before (open columns) and after electrical stimulation (1 Hz for 10 s, filled columns) in the absence (control) and presence of the agents indicated. Each column represents the amount of [3H]NA released in a 2 min collection period and is an average of six to ten observations. Vertical lines show S.E.M. *P < 0-001 compared to net release (stimulated-unstimulated) in control.

Inhibition of [3H1NA release by neuronal blocking agents Guanethidine and related neuronal blockers have long been considered to accumulate specifically in sympathetic neurons through the Uptake 1 system and prevent the release of NA by exerting a local anaesthetic action (Maxwell & Wastila, 1977). We have confirmed the blocking action of guanethidine in neuronal cultures. Figure 3 shows that guanethidine (1 /tM) completely blocked [3H]NA release during stimulation. The effect lasted for several hours even after wash-out of the drug (not shown but see Kirpekar, Wakade, Dixon & Prat, 1969). Figure 3 shows that bretylium (3 ,tM) also caused a complete inhibition of [3H]NA release in cultured neurons. Neither agent significantly affected the release of [31H]NA during the nonstimulation period.

Voltage-clamped Ca2+ currents We used the whole-cell variation of the patch-clamp technique to obtain direct evidence for the modulation of ICa by drugs which facilitated or blocked [3H]NA release in cultured sympathetic neurons. Caesium and TEA were included to block outward K+ currents and TTX to block inward currents through Na+ channels. Figure 4A shows typical ICa evoked by depolarizations from -70 to -20, -10 and 0 mV in a neuron possessing a single neurite. Blocking conduction along the neurite (Fig. 4B, see Methods for details) eliminated the slight latency between currents at the onset of test pulses (arrow 1) and the difference in decay of the tail currents following repolarization (arrows 2 and 3). However, the relative amplitudes of the peak currents elicited at the three test potentials were not affected by the conduction block. There was essentially no difference in the peak Ca2+ current-voltage (I-V) curve (not shown) under the two conditions. The average Ca2+ I-V

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(not shown). We did not observe a rapidly decaying peak in currents evoked from this hyperpolarized holding potential. Figure 6 shows that Ca2+ currents were sensitive to block by cadmium. Cadmium (1-60,M) caused a dose-dependent depression of peak ICa with 92+9 % (n = 7) decrease at 30 /LM.

Cyclic AMP does not alter transmembrane Ca2+ currents Figure 7 shows that when sympathetic neurons were treated with 10 #M-forskolin for 10 min, voltage-elicited ICa was not enhanced. VIP (10 #M for 10 min) had no

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Fig. 7. Agents which facilitate or block neurotransmitter release have no effect on voltageclamped Ca2+ current. Whole-cell Ca2+ currents recorded from the same neuron during control and the indicated drug treatment are shown superimposed. Treatments which facilitated release (upper traces) were 10 ,uM-forskolin or 10 /M-VIP for 10 min. Treatments which blocked release (lower traces) were 1 /LM-guanethidine (15 min) or 3 /Mbretylium (30 min). Horizontal and vertical scales in upper left apply to all traces except bretylium.

effect on ICa. Exposure to the non-hydrolysable cyclic AMP analogue, 8-Br-cyclic AMP (1 mM for 10 min) also failed to affect ICa (not shown). Neuronal blockers do not alter transmembrane Ca2+ currents If guanethidine blocks the release of NA by a selective local anaesthetic action on sympathetic neurons, we anticipate that the drug should have a depressant action on ICa. However, treatment of sympathetic neurons with guanethidine (1 /SM for 15 min) caused no depression of ICa (Fig. 7). Even when neurons were incubated with 3 tMguanethidine for 1 h there was no evidence for ICa depression (462 ± 170 pA, n = 4) compared to untreated cells (477 + 155 pA, n = 23). Figure 7 also shows that bretylium (3 /M for 30 min) had no significant effect on ICa*

Cyclic AMP and neuronal blockers do not effect Na+ and K+ current We considered the possibility that cyclic AMP may reduce outward K+ currents and guanethidine may reduce inward Na+ currents and that these actions could modulate Ca2+ entry indirectly and affect neurotransmitter release. However, forskolin (10 /M) and guanethidine (3/tM) had no effect on outward or inward currents when voltage-clamp experiments were done in the absence of Na+ and K+ current blocking drugs (not shown).

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Intracellular Ca2+ concentration Laser photometry has allowed us to reliably measure [Ca2+]i inside the cell body and the growth cones of sympathetic neurons. [Ca2+]i was calculated using three types of analysis.

Image analysis This mode provided the greatest detail of Ca2+ distribution in the entire region of the neuronal cell body or growth cone under investigation. As shown in Fig. 8, the unstimulated cell body and growth cone exhibited uniform distribution of Ca2+. After electrical stimulation there was a 3- to 7-fold increase in [Ca2+]i in both regions. However, the distribution of Ca2+ in the cell bodies was not uniform. The highest Ca2+ levels were localized in the nucleus, as identified by phase-contrast microscopy (not shown) of the same field. The distribution of Ca2+ in stimulated growth cones

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relatively uniform except for the appearance of random 'hotspots'. Treatment for 10 min with 10 guM-forskolin caused no change in resting Ca2+ levels. The increase

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in [Ca2+]i in the cell body and the growth cone during stimulation was unaffected by forskolin treatment (Fig. 8, 2a-d). Although not shown, VIP also failed to further elevate [Ca2+]i during stimulation in both regions of sympathetic neurons. The most surprising finding was that treatment with guanethidine did not block the increases in [Ca21]i in the cell body and the growth cone during stimulation (Fig. 8, 3a-d). We have also found that the neuronal blocking agent, bretylium, which blocked the evoked release, did not depress the stimulated rise in [Ca2+]i in the cell body and

D. A. PRZYWARA AND OTHERS 2914 growth cone (not shown). In all [Ca2+]i monitoring experiments each cell body or terminal region was used as its own control before drug exposure. The control responses which are not shown were identical to the drug-treated responses shown in Fig. 8. Thus we have found no evidence for modulation of stimulus-evoked increase or decrease of [Ca21]i in the growth cones or cell bodies under conditions which facilitate or block the release of [3H]NA. To ensure that [Ca21]i measurements were sensitive to the drugs we tested the effects of TEA and tetracaine. TEA is known to enhance neuronal Ca21 influx by blocking K+ currents and extending the duration of the nerve action potential (Armstrong & Binstock, 1965). Tetracaine, on the other hand, is a classical local anaesthetic that blocks conduction in neuronal cells and prevents stimulation-induced Ca2` influx (Ritchie & Greengard, 1966). As shown in Fig. 8, TEA (4a-c) caused a marked enhancement of [Ca2+]i whereas tetracaine (5a-c) almost completely blocked the stimulated rise in [Ca2+]i in the cell body and the nerve terminal. Each of the images in Fig. 8 which show a rise in [Ca2+]i also show that the distribution of Ca2+ in the cell body was not uniform. The maximum increase in neuronal Ca2+ was always found in the nucleus, regardless of drug treatment or the method of stimulation. The regional distribution of Ca2+ became apparent only after stimulation and not at rest, and was seen in the ratio images. Line average analysis. Image analysis provided [Ca2+]i distributional information for the whole cell, but data recording required 10 s. Any initial, rapid distribution of Ca2+ would not be observed. Line average analysis was used to observe [Ca21]i changes in a cross-section of the cell at 500 ms time resolution. As shown in Fig. 9, average [Ca2+]i recorded at 500 ms intervals in the cell body (upper traces) and growth cones (lower traces) began to rise immediately with the onset of electrical stimulation. [Ca2+]i increased approximately 3-fold during the stimulation period in both cell bodies and growth cones. Forskolin (10 /kM for 15 min, four experiments) had no effect on resting levels or the stimulated rise of [Ca2+]i in cell bodies (Fig. 9, upper right trace) or in growth cones (not shown). Guanethidine (10 /AM for 30 min, eight experiments) did not inhibit the stimulated increase in [Ca2+]i in growth cones (Fig. 9, lower right trace) or in cell bodies (not shown). Following stimulation, Ca2+ levels in growth cones returned towards resting levels within approximately 10 s while cell body Ca2+ levels fell no more than 20% during the same time period. Point analysis. This mode was used to measure [Ca2+]i at selected points in the cell bodies and growth cones with a 2 ms time resolution. The dynamic Ca2+ responses to stimulation and the various test agents determined by this method were identical to the results obtained by image and line analysis (data not shown).

DISCUSSION

We consider two major findings of the present investigation. First, cyclic AME enhancers facilitate [3H]NA release without further increasing intracellular concentrations of Ca2+ over the levels attained during stimulation. Second, guanethidinelike drugs block [3H]NA release without interfering with the increase in neuronal Ca2+. Both pieces of evidence suggest that release of sympathetic neurotransmitter

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Fig. 9. Agents which facilitate or block neurotransmitter release have no effect on the stimulated rise of [Ca2+]i. Ca2" concentration during 1 Hz electrical stimulation (horizontal bars) in cell body (upper traces) and growth cone (lower traces) in the absence (left) and presence of forskolin (upper right, 10/uM for 15 min) and guanethidine (lower right, 3/M for 30 min). Ca2" responses recorded in the line average mode are from the same cell body (and growth cone) and across the same scan co-ordinates before and after drug treatment. The smoothed plots were obtained by digitally filtering the data using the ACAS software.

can be modulated without affecting the intracellular Ca2+ concentration during stimulation. Recent reports have shown that the modulation of ion channels and the neurotropic actions of forskolin may be independent of the adenylate cyclase-cyclic AMP system (Hoshi, Garber & Aldrich, 1988; Wagoner & Pallotta, 1988; Wakade et al. 1990 b). However, in the present study we establish a direct involvement of cyclic AMP in the modulation of sympathetic transmitter release. The facilitating actions of forskolin and VIP on electrically evoked release of [3H]NA was associated with an increase in cyclic AMP content of sympathetic neurones. 8-Br-cyclic AMP enhanced [3H]NA release and the inactive analogue of forskolin was without effect, suggesting that adenylate cyclase-cyclic AMP was responsible for the facilitation of [3H]NA

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release by forskolin and VIP. These results provide clear and direct evidence for the involvement of cyclic AMP in transmitter facilitation. It has been shown that cyclic AMP enhances the high-voltage-activated, longlasting (L-type) Ca21 channel current in frog sympathetic neurons (Lipscombe, Kongsamut & Tsien, 1989) and neuroblastoma cells (Narahashi, Tsunoo & Yoshii, 1987) as well as in cardiac cells (Reuter, 1983). Our voltage-clamp data indicate that Ca21 channels of chick sympathetic neuronal soma are similar to those found in other cells. However, modulation by cyclic AMP was not observed. The Ca21 current-voltage relation, sensitivity to cadmium and relative lack of inactivation during test pulses are similar to characteristics of L-type Ca 2+ current found in chick sensory neurons (Carbone & Lux, 1984a, b; Fox, Nowycky & Tsien, 1987), rat sympathetic neurons (Schofield & Ikeda, 1988) and neuroblastoma cells (Narahashi et al. 1987). However, embryonic chick sympathetic neurons exhibited peak ICa activation at membrane potentials negative to 0 mV. The ICa activation peaks for L-type Ca2+ currents in other neurons are reported to be between 0 and + 10 mV under similar ionic conditions. Whether this difference in Ca2+ channel activation in chick sympathetic neurons compared to other cells may be related to the lack of cyclic AMP-dependent channel regulation remains to be determined. Any other cyclic AMP-dependent effects on [Ca2+]i, directly on intracellular Ca2+ pools or indirectly through K+ current modulation as found in invertebrate neurons (Castellucci, Kandel, Schwartz, Wilson, Nairn & Greengard, 1980; Klein, Camardo & Kandel, 1982), would have been readily observed in field stimulated neurons monitored by laser photometry. Because growth cones have been reported to be the sites of transmitter release in cultured neurons (Hume, Role & Fischback, 1983; Young & Poo, 1983; Lipscombe, Madison, Poenie, Reuter, Tsien & Tsien, 1988) the possibility exists that local drug actions at the cell body or nerve terminal could account for the differential effects on [3H]NA release and [Ca2+]i. However, we found no difference in drug effects when [Ca2+]i was monitored in growth cones. The kinetics of [Ca2+]i change after stimulation were different in growth cones and cell bodies. Ca2+ was more efficiently removed from the terminal regions (Fig. 9). As a result the same growth cone was able to generate reproducible increases in [Ca2+]i to repetitive stimulations. This property seems to be requisite for a site that is specialized for release of neurotransmitter. Relatively sluggish handling of Ca2+ by the cell body suggests that this region is inefficient in disposition of Ca2' and may not normally participate in the release of transmitter. The difference in Ca2+ handling between cell body and growth cone is most probably due to differences in surface to volume ratio in the two regions. Our findings provide clear evidence that cyclic AMP did not modify influx, efflux or sequestration of Ca2+ to enhance [3H]NA release. These results indicate that cyclic AMP is involved in regulating [3H]NA release at a step down-stream from Ca2+ entry. A site of action for cyclic AMP, distal to Ca2+ entry, has been proposed for the regulation of prolactin release from a pituitary tumour cell line (Frey, Kebabian & Guild, 1986), and for the regulation of catecholamine secretion from the rat adrenal gland (Malhotra et al. 1989). Cyclic AMP may be involved in phosphorylationdependent modulation of [3H]NA release at or near the site of exocytosis. The neuronal secretory vesicle phosphoprotein, synapsin I may act as a substrate for

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cyclic AMP-dependent kinase (see Introduction). Although phosphorylation of synapsin I at the calmodulin kinase II site is responsible for modulating transmitter release in invertebrate neurons, we speculate the cyclic AMP-dependent phosphorylation may be of greater importance in chick sympathetic neurons. We have shown that concentrations of guanethidine and bretylium that block release of [3H]NA have no effect on Ca2+ currents or [Ca2+]i. It has been argued that neuronal blocking agents exert a selective local anaesthetic action and inhibit transmitter release by blocking action potential propagation into the terminal region (Mitchell & Oates, 1970). However, guanethidine, at concentrations which blocked [3H]NA release, had no effect on voltage-clamped, inward or outward currents, and did not block the electrically stimulated increase in [Ca2+]i measured at the growth cone. These results indicate that the neurons had an unaltered electrophysiological response under conditions that blocked transmitter release. To our knowledge, this represents the first example where release of sympathetic transmitter was completely blocked without affecting the increase in neuronal Ca2+ during stimulation. The site of action of the neuronal blocking drugs which, like cyclic AMP, appears to be separate from Ca21 influx, remains to be determined. It is tempting to speculate that the neuronal blockers may alter phosphorylation-dependent events of exocytosis. We have provided evidence that the stimulated rise in [Ca2+]i is greatest in the nuclear area of the cell. The regional increase in Ca2+ cannot be a result of differences in cell thickness, or uneven accumulation of Indo-1 because the ratioing technique eliminates these differences (Grynkiewicz et al. 1985). At present, we do not know the significance of Ca2+ accumulation in the nuclear region. However, a report of a similar phenomenon in bull-frog neurons has appeared since completion of the present work (Hernandez-Cruz, Sala & Adams, 1990). These authors suggest that regional increases in [Ca2+]i could be relevant to Ca2+-regulated nuclear events. In conclusion, we have considered that the role of Ca2+ in exocytosis comprises two events, Ca2+ mobilization and Ca2+ utilization. In the past, agents which enhanced or inhibited exocytotic release of sympathetic transmitter were believed to do so by either increasing or decreasing Ca2+ mobilization. Now we show that the release process was facilitated and blocked without affecting the Ca2+ mobilization step. This suggests that second messengers and drugs may modulate [3H]NA release by affecting Ca2+ utilization. Additional work is necessary to identify the steps of Ca2+ utilization in sympathetic neurons.

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