Br. J. Pharmacol. (1990), 101, 311-318
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The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings 2Jody K. Hirsh, 'Eugene M. Silinsky & 3Carles S. Solsona Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611, U.S.A. 1 The importance of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2 Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.pf) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3 The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4 All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5 The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (3O-100,uM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel. 6 H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250pM). Thus H7 is likely to inhibit PK A in frog motor nerve endings. 7 H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release. 8 The results suggest: (i) that the adenylyl cyclase-cyclic AMP-PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP-PK A system modulates rather than mediates the inhibitory effects of adenosine.
Introduction Adenosine 3': 5'-cyclic monophosphate (cyclic AMP) is a ubiquitous modulator that transduces the activation of specific cell surface receptors into a biological response (Dunwiddie & Hoffer, 1982; North, 1989). At peripheral cholinergic nerve endings, the role of cyclic AMP in coupling presynaptic receptor activation to changes in transmitter release is controversial; cyclic AMP has been suggested to increase (Goldberg & Singer, 1969; Standaert & Dretchen, 1979; Branisteanu et al., 1988; Dryden et al., 1988), decrease (Kuba et al., 1981; Silinsky, 1984), or have no effect (Miyamoto & Breckenridge, 1974) on acetylcholine (ACh) secretion. One possible explanation for these disparate results could be compartmentation of the effects of cyclic AMP in the nerve ending (Harper et al., 1985). Specifically, it has been suggested that increases in cyclic AMP concentration at local submembrane regions could inhibit ACh release (Moskowitz & Puszkin, 1985; Silinsky, 1986) whilst more global increases in cyclic AMP at synaptic vesicles (Moskowitz & Puszkin, 1985; Silinsky, 1985b) or Ca storage sites (Cocks et al., 1984; Silinsky & Vogel, 1986; Harper, 1988) could increase ACh release. ' Author for correspondence. 2 Present address: Department of Physiology, Rush Medical College, 1750 West Harrison Street, Chicago, IL 60612, U.S.A. I Present address: Department of Cellular Biology and Pathology, University of Barcelona, Casanova 143, 08036 Barcelona, Spain.
One objective of this paper is to test the compartmentation hypothesis by comparing the effects of the localized delivery of cyclic AMP to the cytoplasm (using lipid vesicles as a vehicle) with more widespread increases in cytoplasmic cyclic AMP concentrations (using stable, membrane permeant cyclic AMP analogues). Adenosine has been implicated as a negative feedback modulator of ACh release and its receptors are frequently coupled to adenylyl cyclase. Unfortunately, the coupling of adenosine receptor activation to presynaptic adenylyl cyclase is also controversial, with positive coupling, negative coupling, or no coupling through this enzyme being suggested (for review see Silinsky, 1989). Another objective of this paper is to evaluate the contribution of cyclic AMP-dependent processes to the inhibitory effects of adenosine. In this regard, the effects of increases in cyclic AMP concentrations and of inhibition of cyclic AMP-dependent protein kinase (protein kinase A, PK A) on the action of adenosine will be evaluated. H7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) has been found to inhibit PK A, protein kinase C (PK C), and protein kinase G (PK G) with apparent equilibrium dissociation constants (Kis) in the low micromolar range (Hidaka et al., 1984). If it can be shown that H7 fails to alter the presynaptic effects of adenosine in preparations where H7 has been shown to act as a PK A inhibitor, then it is likely that adenosine acts independently of cyclic AMP-dependent protein phosphorylation. The results of this study support the hypothesis that the effects of cyclic AMP are compartmentalized in the nerve
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ending. They also suggest that while the action of adenosine may be modified by changes in cyclic AMP levels, adenosine inhibits ACh release independently of protein kinase A at frog motor nerve endings. Brief accounts of these results have appeared in abstract form (Hirsh et al., 1988; Hirsh & Silinsky, 1989).
Methods Electrophysiological procedures Frog pectoralis proprius nerve-cutaneous pectoris muscle preparations (Rana pipiens) were dissected and superfused with flowing Ringer solution. Conventional electrophysiological techniques for stimulation and recording were employed (see Silinsky, 1984; 1987). Continuous intracellular recordings of endplate potentials (e.p.ps) and miniature endplate potentials (m.e.p.ps) were made from individual endplates, with each fibre serving as its own control. In the majority of instances, experiments were carried out with the assistance of an LSI 11/73 minicomputer (Cambridge Digital) and 125 kHz 14 bit A/D-12 bit D/A converter on line with a hard copy of the digitalized traces being made on an XY plotter (Hewlett Packard 7470A). In a few experiments, e.p.ps were averaged by a computer of average transients (Nicolet) in which case the average was displayed on a pen recorder or XY plotter. In most experiments, the frequency of m.e.p.ps (m.e.p.p.f) and m.e.p.p. amplitudes were determined by an automated analysis using the LSI computer in conjunction with a circulating buffer and a FORTRAN programme that uses an algorithm based upon the initial slope of the rising phase to detect an event ('mepp-o-matic'). The m.e.p.ps were first recorded on magnetic tape (Indec IR2 Instrumentation Recorder) before analysis by minicomputer. In some experiments, ACh potentials were produced by applying puffs of ACh (100-500pM dissolved in Ringer) from a 1-3 MQ microelectrode connected to a source of pressurized gas (Picospritzer II, General Valve Corporation).
been used successfully in the past to deliver this membraneimpermeable nucleotide to the cytoplasm of living cells (Papahadjopoulos et al., 1974).
Statistical methods Statistical procedures were identical to those described previously (see Silinsky, 1984; 1987). Statistically significant differences were generally observed at P 3
2 0
CU
E 01
Control
H7
H7
Adenosine
b
8r
C: a)
0
4
a) If
i 2 a)
Control Adenosine
Figure 6 H7 does not alter the effect of adenosine. Solutions were sequentially superfused from left to right as follows: Control (0.35 mM Ca 3.0mM Mg Ringer); 60,UM H7; 60,M H7 and 50pM adenosine; 60,UM H7. Each column represents the m (a, n = 256 to 640 sweeps) or the mean miniature endplate potential frequency (m.e.p.p.f) (b, n = 30 to 60) at a time when the superfusion solution had attained a steadystate effect. Note that adenosine produced a typical effect, specifically a 58.1% decline in m, and a 59.9% reduction in m.e.p.p.f ence of H7. release (m) vs treatment; n = 384-448 sweeps,
in the pres1.0 Hz.
The results demonstrate that CPT cyclic AMP, a stable lipophilic cyclic AMP analogue with a high potency as a PK A activator exclusively produces increases in ACh, whilst the local delivery of the membrane impermeant natural messenger cyclic AMP via liposomes inhibits ACh release. db Cyclic AMP, a lipophilic analogue with low potency as a PK A activator, produced smaller increases in evoked ACh release and on occasion may even have inhibited transmitter secretion. The most plausible explanation for these results is that multiple cellular compartments for cyclic AMP exist within the nerve terminal (Silinsky & Vogel, 1986; see also Dunwiddie & Hoffer, 1982; Phillis & Barraco, 1985). Compartmentation of second messenger action is an established phenomenon (Harper et al., 1985; Harper, 1988) and has even been suggested to explain the complex effects of cyclic nucleotides at nerve endings (Moskowitz & Puszkin, 1985; Silinsky & Vogel, 1986). Specifically, it has been suggested that local submembraneous increases in cyclic AMP (as would be produced by cyclic AMP in liposomes) inhibit ACh release whilst more global increases in cyclic AMP concentrations (such as those produced by lipophilic cyclic AMP analogues) phosphorylate vesicular proteins or Ca translocation sites and stimulate secretion. Support for the hypothesis of local delivery of liposomal contents just beneath the plasma membrane in secretory cells may be found in a number of different studies. Ca delivered in liposomes evokes highly-localized exocytotic responses at the sites of liposome fusion in mast cells (Theoharides & Douglas, 1978). Moreover, Ca- and Srcontaining liposomes produce selective effects on m relative to spontaneous ACh release (Mellow et al., 1982) and studies with the Ca buffer BAPTA suggest m is dependent on local, submembraneous Ca rather than bulk, cytoplasmic Ca (Kijima & Tanabe, 1988). With respect to the stimulating effects of cyclic AMP analogues, it has been suggested that phosphorylation of proteins
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associated with synaptic vesicles (e.g Synapsin I) increase the availability of the transmitter for release (Silinsky, 1985a, b; Miller, 1985; Bahler & Greengard, 1987; Moskowitz & Puszkin, 1985; Harper, 1988; Dryden et al., 1988). These stimulating effects could also arise secondarily as a result of increases in cellular Ca levels caused by cyclic AMP accumulation (Moskowitz & Puszkin, 1985; Tomlinson et al., 1985; Silinsky & Vogel, 1986; Harper, 1988). The excitatory actions of cyclic AMP analogues are unlikely to occur exclusively by the stimulation of Ca entry through voltage-gated Ca channels; excitatory effects of these analogues on m.e.p.p1 were observed in Ca-free solution. With respect to decreases in ACh release, it has been suggested that phosphorylation of a Ca binding protein intimately associated with the secretory apparatus might yield a decreased affinity for Ca and thus inhibit release (see Silinsky, 1984; 1986 for discussion of this mechanism). Alternatively, cyclic AMP could stimulate Ca uptake into cellular storage sites near the plasma membrane; cyclic AMP-driven Ca uptake has been found in lysed brain synaptosomes (MekhailIshak et al., 1987). In this regard, cyclic AMP-dependent phosphorylation of inositol trisphosphate receptors has been found to decrease Ca release (Supattapone et al., 1988). Regardless of the precise mechanism, the suggestion of opposing effects of cyclic AMP at different parts of the secretory apparatus appears to provide the simplest explanation for the results presented herein. Specifically, a balance between phosphorylation near the membrane and phosphorylation of more distal sites appears to determine whether a cyclic AMP analogue will produce excitation or inhibition. Based upon these results and previous results with PDE inhibitors (Silinsky, 1984; Silinsky & Vogel, 1987; Dryden et al., 1988), it is likely that the more distal of these mechanisms, i.e. the one responsible for the excitatory effects of cyclic AMP, possesses the greatest capacity to influence the secretory process. When compared to the results of others using lipophilic cyclic AMP analogues, the present results generally agree with those of Dryden et al. (1988) who obtained stimulating effects of 8-Br-cyclic AMP on mouse motor nerve terminals. The stimulating effects of db cyclic AMP in the present study in the frog also agree with most previous studies in the frog (Branisteanu et al., 1988), the cat (Standaert & Dretchen, 1979) and the rat (Goldberg & Singer, 1969), but do not accord with other studies in the rat (Miyamoto & Breckenridge, 1974) or the mouse (Dryden et al., 1988) where no effects were demonstrated. It should be noted that biphasic effects of db cyclic AMP, an initial inhibition during exposure to this analogue and a rebound increase in ACh release after washing with drug-free solution, were consistently observed in bullfrog sympathetic ganglia (Kuba et al., 1981). We also observed such an effect in two of the six experiments with db cyclic AMP. Biological variability and species specificity could explain why DB cyclic AMP fails to produce uniform effects at peripheral cholinergic nerve endings.
Cyclic AMP and the interactions with adenosine The observations that local increases in cyclic AMP mimic the inhibitory effects of adenosine, and that cyclic AMP and its congeners decrease the inhibitory effects of adenosine, support the suggestion that adenosine could be increasing local cyclic AMP levels to inhibit ACh release (Silinsky, 1984). Because this adenosiqe receptor is apparently linked to a pertussis toxin-sensitive G-protein (Silinsky et al., 1989; Chen et al., 1989), and blockade of PK A by H7 does not alter the effects of adenosine, it appears more likely that phosphorylation via PK A modulates rather than mediates the actions of adenosine.
What are some phosphorylation sites that may impair the action of adenosine? One possibility is that phosphorylation of the adenosine receptor itself could decrease the effects of adenosine in a manner similar to the heterologous desensitization produced by increases in cellular cyclic AMP levels
(see Huganir & Greengard, 1987 for a review). Alternatively, nerve terminal calcium binding proteins could be phosphorylated and reduce the effectiveness of adenosine (see above). Whatever the mechanism, it is unlikely to be related to extracellular effects of cyclic AMP analogues on adenosine receptors as: (i) the effects of cyclic AMP in liposomes were not blocked by high concentrations of the adenosine receptor blocker, theophylline (Figure 2); (ii) in the experiment shown in Figure 3, CPT cyclic AMP antagonized the effect of adenosine only on spontaneous ACh release and not evoked ACh secretion (see figure legend), (iii) in several experiments in which db cyclic AMP, inhibited the action of adenosine, this analogue was washed out of the bathing fluid before the addition of adenosine, and (iv) cyclic AMP in liposomes inhibited the action of adenosine under conditions in which cyclic AMP liposomes in themselves did not alter ACh release (n = 1, data not shown).
Effects of protein kinase inhibition With respect to the control of presynaptic function by protein kinases, consistent changes in evoked and spontaneous ACh release failed to occur in the presence of H7. This suggests that phosphorylation by H7-sensitive kinases does not play a substantial role in ACh release in the unperturbed system. Modulation of ACh release by these kinases can still occur, as shown by the effects of cyclic AMP derivatives and phorbol esters. With respect to the action of adenosine, the results of this study suggest that adenosine inhibits ACh release from frog motor nerve endings independently of protein phosphorylation by any kinases which are affected by H7. H7 is likely to be inhibiting PK A in this species, as it can prevent increases in spontaneous ACh release produced by the potent PK A stimulator CPT cyclic AMP. As H7 has been found to prevent TPA-induced increases in spontaneous ACh release (Branisteanu et al., 1988; Caratsch et al., 1988), PK C also appears to be inhibited under conditions where adenosine exerts its typical effects on ACh release. A comparison of our results with previous data in the literature reveals considerable controversy. Chen et al. (1989) showed that 501M H7 blocked the effects of 2-chloroadenosine and decreased basal levels of spontaneous ACh release in the mouse phrenic nerve-hemidiaphragm. It is possible that species heterogeneity could account for this observation. Most puzzling, however, is the result of Branisteanu et al. (1988) where the presence of 10pM H7 converted the effect of 50pM adenosine into stimulation of spontaneous ACh release in cutaneous pectoris nerve-muscle preparations from Rana ridibunda. We have no reasonable explanation for this discrepancy, especially since stimulating effects of adenosine on ACh release are believed to be mediated by stimulation of adenylyl cyclase (see Silinsky, 1989 for a review). However, in partial accordance with our results Branisteanu et al. (1988) showed that H7 alone did not alter the m.e.p.p. frequency. With respect to the postjunctional action of H7, the apparent blockade of the nicotinic receptor/ionic channel by this drug has not been widely demonstrated in neuromuscular
preparations (Caratsch et al., 1988; Branisteanu et al., 1988; Chen et al., 1989; but see Sebastiao, 1989). It is of interest that H7 decreases the mean open time of single channel currents induced by ACh at nicotinic receptors expressed in Xenopus oocytes (Reuhl et al., 1989). These effects at nicotinic receptors are likely to be produced by channel block (Peper et al., 1982), although the precise kinetic model awaits studies under voltage clamp and at the level of single ionic channels. Regardless of mechanism, these postjunctional effects could potentially lead to the erroneous conclusion that ACh release is decreased in the presence of H7. This is especally true if the channel block is use-dependent and repetitive nerve stimulation is employed. In conclusion, it appears that the cyclic AMP-PK A system is compartmentalized at motor nerve endings and modulates the effects of adenosine. Adenosine, however, inhibits ACh
CYCLIC AMP-ADENOSINE INTERACTIONS IN NERVE ENDINGS
release from motor nerve endings by a mechanism independent of PK A, PK C, and possibly other kinases in the frog. These results do not exclude the remote possibility that cyclic AMP may mediate the effects of adenosine independently of protein phosphorylation. However, a more conventional interpretation is that the adenosine receptor at motor nerve endings is coupled directly through a G protein to its cellular effector, as has been shown for Ca channels and potassium
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channels in some neuronal soma (see Silinsky, 1989 for review). At frog motor nerve endings, this cellular effector is likely to be a Ca-sensitive component of the secretory apparatus (Silinsky, 1986; Silinsky et al., 1989). This work was supported by a research grant from the U.S. Public Health Service (NS12782). J.K.H. was also supported by a predoctoral fellowship from the Lucille P. Markey Charitable Trust.
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STEINER, J. & SNYDER, S.H. (1988). Cyclic AMP-dependent phos(Received March i9, 1990 Revised May 23, 1990 Accepted May 24, 1990)
(D Macmillan Press Ltd, 1990
The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings 2Jody K. Hirsh, 'Eugene M. Silinsky & 3Carles S. Solsona Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611, U.S.A. 1 The importance of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2 Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.pf) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3 The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4 All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5 The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (3O-100,uM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel. 6 H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250pM). Thus H7 is likely to inhibit PK A in frog motor nerve endings. 7 H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release. 8 The results suggest: (i) that the adenylyl cyclase-cyclic AMP-PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP-PK A system modulates rather than mediates the inhibitory effects of adenosine.
Introduction Adenosine 3': 5'-cyclic monophosphate (cyclic AMP) is a ubiquitous modulator that transduces the activation of specific cell surface receptors into a biological response (Dunwiddie & Hoffer, 1982; North, 1989). At peripheral cholinergic nerve endings, the role of cyclic AMP in coupling presynaptic receptor activation to changes in transmitter release is controversial; cyclic AMP has been suggested to increase (Goldberg & Singer, 1969; Standaert & Dretchen, 1979; Branisteanu et al., 1988; Dryden et al., 1988), decrease (Kuba et al., 1981; Silinsky, 1984), or have no effect (Miyamoto & Breckenridge, 1974) on acetylcholine (ACh) secretion. One possible explanation for these disparate results could be compartmentation of the effects of cyclic AMP in the nerve ending (Harper et al., 1985). Specifically, it has been suggested that increases in cyclic AMP concentration at local submembrane regions could inhibit ACh release (Moskowitz & Puszkin, 1985; Silinsky, 1986) whilst more global increases in cyclic AMP at synaptic vesicles (Moskowitz & Puszkin, 1985; Silinsky, 1985b) or Ca storage sites (Cocks et al., 1984; Silinsky & Vogel, 1986; Harper, 1988) could increase ACh release. ' Author for correspondence. 2 Present address: Department of Physiology, Rush Medical College, 1750 West Harrison Street, Chicago, IL 60612, U.S.A. I Present address: Department of Cellular Biology and Pathology, University of Barcelona, Casanova 143, 08036 Barcelona, Spain.
One objective of this paper is to test the compartmentation hypothesis by comparing the effects of the localized delivery of cyclic AMP to the cytoplasm (using lipid vesicles as a vehicle) with more widespread increases in cytoplasmic cyclic AMP concentrations (using stable, membrane permeant cyclic AMP analogues). Adenosine has been implicated as a negative feedback modulator of ACh release and its receptors are frequently coupled to adenylyl cyclase. Unfortunately, the coupling of adenosine receptor activation to presynaptic adenylyl cyclase is also controversial, with positive coupling, negative coupling, or no coupling through this enzyme being suggested (for review see Silinsky, 1989). Another objective of this paper is to evaluate the contribution of cyclic AMP-dependent processes to the inhibitory effects of adenosine. In this regard, the effects of increases in cyclic AMP concentrations and of inhibition of cyclic AMP-dependent protein kinase (protein kinase A, PK A) on the action of adenosine will be evaluated. H7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) has been found to inhibit PK A, protein kinase C (PK C), and protein kinase G (PK G) with apparent equilibrium dissociation constants (Kis) in the low micromolar range (Hidaka et al., 1984). If it can be shown that H7 fails to alter the presynaptic effects of adenosine in preparations where H7 has been shown to act as a PK A inhibitor, then it is likely that adenosine acts independently of cyclic AMP-dependent protein phosphorylation. The results of this study support the hypothesis that the effects of cyclic AMP are compartmentalized in the nerve
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ending. They also suggest that while the action of adenosine may be modified by changes in cyclic AMP levels, adenosine inhibits ACh release independently of protein kinase A at frog motor nerve endings. Brief accounts of these results have appeared in abstract form (Hirsh et al., 1988; Hirsh & Silinsky, 1989).
Methods Electrophysiological procedures Frog pectoralis proprius nerve-cutaneous pectoris muscle preparations (Rana pipiens) were dissected and superfused with flowing Ringer solution. Conventional electrophysiological techniques for stimulation and recording were employed (see Silinsky, 1984; 1987). Continuous intracellular recordings of endplate potentials (e.p.ps) and miniature endplate potentials (m.e.p.ps) were made from individual endplates, with each fibre serving as its own control. In the majority of instances, experiments were carried out with the assistance of an LSI 11/73 minicomputer (Cambridge Digital) and 125 kHz 14 bit A/D-12 bit D/A converter on line with a hard copy of the digitalized traces being made on an XY plotter (Hewlett Packard 7470A). In a few experiments, e.p.ps were averaged by a computer of average transients (Nicolet) in which case the average was displayed on a pen recorder or XY plotter. In most experiments, the frequency of m.e.p.ps (m.e.p.p.f) and m.e.p.p. amplitudes were determined by an automated analysis using the LSI computer in conjunction with a circulating buffer and a FORTRAN programme that uses an algorithm based upon the initial slope of the rising phase to detect an event ('mepp-o-matic'). The m.e.p.ps were first recorded on magnetic tape (Indec IR2 Instrumentation Recorder) before analysis by minicomputer. In some experiments, ACh potentials were produced by applying puffs of ACh (100-500pM dissolved in Ringer) from a 1-3 MQ microelectrode connected to a source of pressurized gas (Picospritzer II, General Valve Corporation).
been used successfully in the past to deliver this membraneimpermeable nucleotide to the cytoplasm of living cells (Papahadjopoulos et al., 1974).
Statistical methods Statistical procedures were identical to those described previously (see Silinsky, 1984; 1987). Statistically significant differences were generally observed at P 3
2 0
CU
E 01
Control
H7
H7
Adenosine
b
8r
C: a)
0
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a) If
i 2 a)
Control Adenosine
Figure 6 H7 does not alter the effect of adenosine. Solutions were sequentially superfused from left to right as follows: Control (0.35 mM Ca 3.0mM Mg Ringer); 60,UM H7; 60,M H7 and 50pM adenosine; 60,UM H7. Each column represents the m (a, n = 256 to 640 sweeps) or the mean miniature endplate potential frequency (m.e.p.p.f) (b, n = 30 to 60) at a time when the superfusion solution had attained a steadystate effect. Note that adenosine produced a typical effect, specifically a 58.1% decline in m, and a 59.9% reduction in m.e.p.p.f ence of H7. release (m) vs treatment; n = 384-448 sweeps,
in the pres1.0 Hz.
The results demonstrate that CPT cyclic AMP, a stable lipophilic cyclic AMP analogue with a high potency as a PK A activator exclusively produces increases in ACh, whilst the local delivery of the membrane impermeant natural messenger cyclic AMP via liposomes inhibits ACh release. db Cyclic AMP, a lipophilic analogue with low potency as a PK A activator, produced smaller increases in evoked ACh release and on occasion may even have inhibited transmitter secretion. The most plausible explanation for these results is that multiple cellular compartments for cyclic AMP exist within the nerve terminal (Silinsky & Vogel, 1986; see also Dunwiddie & Hoffer, 1982; Phillis & Barraco, 1985). Compartmentation of second messenger action is an established phenomenon (Harper et al., 1985; Harper, 1988) and has even been suggested to explain the complex effects of cyclic nucleotides at nerve endings (Moskowitz & Puszkin, 1985; Silinsky & Vogel, 1986). Specifically, it has been suggested that local submembraneous increases in cyclic AMP (as would be produced by cyclic AMP in liposomes) inhibit ACh release whilst more global increases in cyclic AMP concentrations (such as those produced by lipophilic cyclic AMP analogues) phosphorylate vesicular proteins or Ca translocation sites and stimulate secretion. Support for the hypothesis of local delivery of liposomal contents just beneath the plasma membrane in secretory cells may be found in a number of different studies. Ca delivered in liposomes evokes highly-localized exocytotic responses at the sites of liposome fusion in mast cells (Theoharides & Douglas, 1978). Moreover, Ca- and Srcontaining liposomes produce selective effects on m relative to spontaneous ACh release (Mellow et al., 1982) and studies with the Ca buffer BAPTA suggest m is dependent on local, submembraneous Ca rather than bulk, cytoplasmic Ca (Kijima & Tanabe, 1988). With respect to the stimulating effects of cyclic AMP analogues, it has been suggested that phosphorylation of proteins
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associated with synaptic vesicles (e.g Synapsin I) increase the availability of the transmitter for release (Silinsky, 1985a, b; Miller, 1985; Bahler & Greengard, 1987; Moskowitz & Puszkin, 1985; Harper, 1988; Dryden et al., 1988). These stimulating effects could also arise secondarily as a result of increases in cellular Ca levels caused by cyclic AMP accumulation (Moskowitz & Puszkin, 1985; Tomlinson et al., 1985; Silinsky & Vogel, 1986; Harper, 1988). The excitatory actions of cyclic AMP analogues are unlikely to occur exclusively by the stimulation of Ca entry through voltage-gated Ca channels; excitatory effects of these analogues on m.e.p.p1 were observed in Ca-free solution. With respect to decreases in ACh release, it has been suggested that phosphorylation of a Ca binding protein intimately associated with the secretory apparatus might yield a decreased affinity for Ca and thus inhibit release (see Silinsky, 1984; 1986 for discussion of this mechanism). Alternatively, cyclic AMP could stimulate Ca uptake into cellular storage sites near the plasma membrane; cyclic AMP-driven Ca uptake has been found in lysed brain synaptosomes (MekhailIshak et al., 1987). In this regard, cyclic AMP-dependent phosphorylation of inositol trisphosphate receptors has been found to decrease Ca release (Supattapone et al., 1988). Regardless of the precise mechanism, the suggestion of opposing effects of cyclic AMP at different parts of the secretory apparatus appears to provide the simplest explanation for the results presented herein. Specifically, a balance between phosphorylation near the membrane and phosphorylation of more distal sites appears to determine whether a cyclic AMP analogue will produce excitation or inhibition. Based upon these results and previous results with PDE inhibitors (Silinsky, 1984; Silinsky & Vogel, 1987; Dryden et al., 1988), it is likely that the more distal of these mechanisms, i.e. the one responsible for the excitatory effects of cyclic AMP, possesses the greatest capacity to influence the secretory process. When compared to the results of others using lipophilic cyclic AMP analogues, the present results generally agree with those of Dryden et al. (1988) who obtained stimulating effects of 8-Br-cyclic AMP on mouse motor nerve terminals. The stimulating effects of db cyclic AMP in the present study in the frog also agree with most previous studies in the frog (Branisteanu et al., 1988), the cat (Standaert & Dretchen, 1979) and the rat (Goldberg & Singer, 1969), but do not accord with other studies in the rat (Miyamoto & Breckenridge, 1974) or the mouse (Dryden et al., 1988) where no effects were demonstrated. It should be noted that biphasic effects of db cyclic AMP, an initial inhibition during exposure to this analogue and a rebound increase in ACh release after washing with drug-free solution, were consistently observed in bullfrog sympathetic ganglia (Kuba et al., 1981). We also observed such an effect in two of the six experiments with db cyclic AMP. Biological variability and species specificity could explain why DB cyclic AMP fails to produce uniform effects at peripheral cholinergic nerve endings.
Cyclic AMP and the interactions with adenosine The observations that local increases in cyclic AMP mimic the inhibitory effects of adenosine, and that cyclic AMP and its congeners decrease the inhibitory effects of adenosine, support the suggestion that adenosine could be increasing local cyclic AMP levels to inhibit ACh release (Silinsky, 1984). Because this adenosiqe receptor is apparently linked to a pertussis toxin-sensitive G-protein (Silinsky et al., 1989; Chen et al., 1989), and blockade of PK A by H7 does not alter the effects of adenosine, it appears more likely that phosphorylation via PK A modulates rather than mediates the actions of adenosine.
What are some phosphorylation sites that may impair the action of adenosine? One possibility is that phosphorylation of the adenosine receptor itself could decrease the effects of adenosine in a manner similar to the heterologous desensitization produced by increases in cellular cyclic AMP levels
(see Huganir & Greengard, 1987 for a review). Alternatively, nerve terminal calcium binding proteins could be phosphorylated and reduce the effectiveness of adenosine (see above). Whatever the mechanism, it is unlikely to be related to extracellular effects of cyclic AMP analogues on adenosine receptors as: (i) the effects of cyclic AMP in liposomes were not blocked by high concentrations of the adenosine receptor blocker, theophylline (Figure 2); (ii) in the experiment shown in Figure 3, CPT cyclic AMP antagonized the effect of adenosine only on spontaneous ACh release and not evoked ACh secretion (see figure legend), (iii) in several experiments in which db cyclic AMP, inhibited the action of adenosine, this analogue was washed out of the bathing fluid before the addition of adenosine, and (iv) cyclic AMP in liposomes inhibited the action of adenosine under conditions in which cyclic AMP liposomes in themselves did not alter ACh release (n = 1, data not shown).
Effects of protein kinase inhibition With respect to the control of presynaptic function by protein kinases, consistent changes in evoked and spontaneous ACh release failed to occur in the presence of H7. This suggests that phosphorylation by H7-sensitive kinases does not play a substantial role in ACh release in the unperturbed system. Modulation of ACh release by these kinases can still occur, as shown by the effects of cyclic AMP derivatives and phorbol esters. With respect to the action of adenosine, the results of this study suggest that adenosine inhibits ACh release from frog motor nerve endings independently of protein phosphorylation by any kinases which are affected by H7. H7 is likely to be inhibiting PK A in this species, as it can prevent increases in spontaneous ACh release produced by the potent PK A stimulator CPT cyclic AMP. As H7 has been found to prevent TPA-induced increases in spontaneous ACh release (Branisteanu et al., 1988; Caratsch et al., 1988), PK C also appears to be inhibited under conditions where adenosine exerts its typical effects on ACh release. A comparison of our results with previous data in the literature reveals considerable controversy. Chen et al. (1989) showed that 501M H7 blocked the effects of 2-chloroadenosine and decreased basal levels of spontaneous ACh release in the mouse phrenic nerve-hemidiaphragm. It is possible that species heterogeneity could account for this observation. Most puzzling, however, is the result of Branisteanu et al. (1988) where the presence of 10pM H7 converted the effect of 50pM adenosine into stimulation of spontaneous ACh release in cutaneous pectoris nerve-muscle preparations from Rana ridibunda. We have no reasonable explanation for this discrepancy, especially since stimulating effects of adenosine on ACh release are believed to be mediated by stimulation of adenylyl cyclase (see Silinsky, 1989 for a review). However, in partial accordance with our results Branisteanu et al. (1988) showed that H7 alone did not alter the m.e.p.p. frequency. With respect to the postjunctional action of H7, the apparent blockade of the nicotinic receptor/ionic channel by this drug has not been widely demonstrated in neuromuscular
preparations (Caratsch et al., 1988; Branisteanu et al., 1988; Chen et al., 1989; but see Sebastiao, 1989). It is of interest that H7 decreases the mean open time of single channel currents induced by ACh at nicotinic receptors expressed in Xenopus oocytes (Reuhl et al., 1989). These effects at nicotinic receptors are likely to be produced by channel block (Peper et al., 1982), although the precise kinetic model awaits studies under voltage clamp and at the level of single ionic channels. Regardless of mechanism, these postjunctional effects could potentially lead to the erroneous conclusion that ACh release is decreased in the presence of H7. This is especally true if the channel block is use-dependent and repetitive nerve stimulation is employed. In conclusion, it appears that the cyclic AMP-PK A system is compartmentalized at motor nerve endings and modulates the effects of adenosine. Adenosine, however, inhibits ACh
CYCLIC AMP-ADENOSINE INTERACTIONS IN NERVE ENDINGS
release from motor nerve endings by a mechanism independent of PK A, PK C, and possibly other kinases in the frog. These results do not exclude the remote possibility that cyclic AMP may mediate the effects of adenosine independently of protein phosphorylation. However, a more conventional interpretation is that the adenosine receptor at motor nerve endings is coupled directly through a G protein to its cellular effector, as has been shown for Ca channels and potassium
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channels in some neuronal soma (see Silinsky, 1989 for review). At frog motor nerve endings, this cellular effector is likely to be a Ca-sensitive component of the secretory apparatus (Silinsky, 1986; Silinsky et al., 1989). This work was supported by a research grant from the U.S. Public Health Service (NS12782). J.K.H. was also supported by a predoctoral fellowship from the Lucille P. Markey Charitable Trust.
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STEINER, J. & SNYDER, S.H. (1988). Cyclic AMP-dependent phos(Received March i9, 1990 Revised May 23, 1990 Accepted May 24, 1990)
