Clinical and Experimental Pharmacology and Physiology (1992) 19,631-643
PREJUNCTIONAL ACTIONS OF TACRINE ON AUTONOMIC NEUROEFFECTOR TRANSMISSION IN RABBIT ISOLATED PULMONARY ARTERY AND RAT ISOLATED ATRIA Maurizio E. Fabiani, Peter Kabo and David F. Story Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia (Received 7 January 1992; revision received 29 May 1992)
SUMMARY 1. This study investigated the effects of tacrine (1,2,3,4-tetrahydr0-9-aminoacridine) on the resting and stimulation-induced (SI) release of radioactive substances from isolated preparations of rat atria and rabbit pulmonary artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline, and from rat atrial preparations in which cholinergic transmitter stores had been labelled with [3H]-acetylcholine. In addition, the effect of tacrine on the uptake of [3H]noradrenaline by noradrenergic nerves in rat atria was determined. 2. Tacrine produced concentration-dependent increases in the resting efflux of radioactivity from both the [3H]-noradrenaline-loaded artery and atrial preparations. Blockade of neuronal amine transport with desipramine reduced the release of radioactivity evoked by tacrine from atria but not that evoked from artery preparations. Inhibition of monoamine oxidase by pargyline pretreatment markedly reduced the tacrine-evoked release of radioactivity in both atrial and artery preparations. 3. The radioactivity released from [3H]-noradrenaline-labelled rat atrial preparations by 30 pmol/ L tacrine consisted entirely of the deaminated metabolite [3H]-DOPEG. The evoked release of [3H]-DOPEG from atria was reduced by approximately 50% by desipramine (1 pmol/L). When atrial monoamine oxidase had been inhibited by pargyline treatment in vivo and in vitro, 30 pmol/ L tacrine evoked the release of [3H]-noradrenaline instead of [3H]-DOPEG. However, the amounts of [3H]-noradrenaline released by tacrine when monoamine oxidase was inhibited were only about 25% of the amounts of [3H]-DOPEG released in untreated atria. 4. Tacrine, in concentrations of I and 10 pmol/L, enhanced the release of radioactivity evoked by field stimulation of [3H]-noradrenaline-loaded rabbit pulmonary artery preparations. This effect was unaltered by desipramine or pretreatment with pargyline. However, in artery preparations pretreated with pargyline, a high concentration of tacrine (100 pmol/ L) markedly reduced SI efflux. In contrast to the findings with artery preparations, tacrine (1 -30 pmol/ L) did not alter SI efflux in rat atrial preparations. 5. It is concluded that tacrine displaces noradrenaline from intraneuronal transmitter stores of sympathetically-innervatedtissues, and that the displaced amine is totally metabolized by monoamine oxidase before leaving the nerve terminals. When deamination of neuronal cytoplasmic noradrenaline is prevented, only a portion of the noradrenaline displaced from storage vesicles passes to the extracellular space. It is likely that the transfer of cytoplasmic noradrenaline out of the terminals is limited by the activity of the amine transport mechanism.
Correspondence: Associate Professor David F. Story, Department of Pharmacology, IJniversityof Melbourne,Parkville, Vic. 3052, Australia.
632
M. E. Fabiani et al. 6. Tacrine, in concentrations of 30 and 100 pmol/L, reduced the uptake radioactivity by rat atria incubated for 5 min periods in [3H]-noradrenaline to approximately 83 and 26%, respectively, of control uptake. Desipramine was much more potent than tacrine in inhibiting [3H]-noradrenaline uptake: 1 pmol/L desipramine reduced the uptake radioactivity to approximately 18% of the control. 7. Tacrine (30 pmol/ L) did not alter the resting efflux of radioactivity from [3H]-acetylcholinelabelled rat atrial preparations, but it reduced the efflux of radioactivity evoked by stimulation of intramural cholinergic nerves. The inhibition of SI efflux in the [3H]-acetylcholine-labelled atria may have been mediated by acetylcholine that had accumulated as a consequence of the anticholinesterase activity of tacrine at cholinergic nerve terminals. Key words: cholinergic transmission, noradrenergic transmission, potassium channel antagonists, rabbit pulmonary artery, rat atria, tacrine.
INTRODUCTION There has been renewed interest in the anticholinesterase drug tacrine ( 1,2,3,4-tetrahydro-9-aminoacridine; THA) following reports of its beneficial effects in Alzheimer's disease (Summers et al. 1986; Becker k Giacobini 1988; Gauthier et al. 1988; Nyback et al. 1988). The anticholinesterase activity of tacrine was first reported by Shaw and Bentley (1953). Subsequently, it was shown to inhibit both acetycholinesterase and pseudocholinesterase (Heilbronn 1961; Ho & Freeman 1965). In view of the cholinergic deficit of central neurones in Alzheimer's disease (Davis & Maloney 1976; Davis & Mohs 1986), it might be assumed that any therapeutic effect of tacrine in Alzheimer's disease is dependent on its anticholinesterase activity. However, other anticholinesterase drugs such as physostigmine (Stern et al. 1987), muscarinic agonists such as pilocarpine (Caine 1980; Bruno et al. 1986) and arecoline (Tariot 1988), and the acetylcholineprecursor choline (Becker & Giacobini 1988) produce no apparent improvement in the disease. Thus, an action of tacrine other than or in addition to the inhibition of cholinesterase enzymes may be involved in any beneficial effect of the drug in Alzheimer's disease. Indeed Summers et al. (1986) suggested that potassium channelblocking activity may play a role in the putative therapeutic effect of tacrine in Alzheimer's disease. Tacrine has structural similarities to the sodium channel-blocking drug 9-aminoacridine (Yeh 1979; Yamamoto & Yeh 1984) and also to the potassium channel-blockingdrug 4-aminopyridine (Soni & Kam 1982; Wesseling & Agoston 1984; Davidson et al. 1988). Moreover, tacrine has been reported to block sodium channels in myelinated axons of the toad Xenopus laevis (Elinder et al. 1989) and in the giant axons of the worm Myxicola (Schauf & Sattin 1987).
In addition, several studies have shown that tacrine blocks potassium channels in a range of excitable cells, including guinea-pig ventricular myocytes (Osterrieder 1987) and atria (Freeman et al. 1988)' rat hippocampal neurones (Rogawski 1987; Halliwell & Grove 1989), and neurones of Xenopus laevis (Elinder et al. 1989), Myxicola (Schauf & Sattin 1987) and of the snail Lyrnnea stagnalis (Drukarch et al. 1987). There is also evidence to suggest that tacrine may have some blocking activity at voltage-dependent Caz+ channels (Elinder et al. 1989). Relatively little is known about the effects of tacrine on neurotransmitter release. In view of the reports of its beneficial effects in Alzheimer's disease, the interaction of tacrine with neurotransmitter systems is worthy of further investigation. The present study was undertaken to investigate the effects of tacrine on noradrenergic and cholinergic autonomic neuroeffector transmission. The effects of tacrine on symphathetic noradrenergic transmission were investigated in isolated preparations of rabbit pulmonary artery and rat atria in which noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. Its effects on cholinergic transmission were investigated in rat atrial prepara#ionsin which the cholinergic transmitter stores had been YhdiolabetIed with [3H+acetylcholine.
METHODS Rabbit pulmonary artery preparation Rabbits of either sex (2-4 kg) were sacrificed a blow to the neck and exsanguinated. The chest was opened and the heart rapidly removed. A segment of the common pulmonary artery was dissected free and cut
Effects of tacrine on neuroeffector transmission
into two spiral strips approximately 20 mm long and 2 mm wide. The artery strips were incubated for 20 rnin with ['HI-noradrenaline (10 pCi/mL, 0.67 pmol/ L), contained in 1 mL of physiological salt solution (PSS) in a jacketed organ bath. The PSS had the following composition (mmol/L): NaCl ( I 18); KCl(4.7); CaC12 (2.5); MgS04 (0.45); NaHC03 (25); KH2P04 (1.03); and D-( +)-glucose (1 I. 1). Disodium edetate (0.067 mmol/ L) and ascorbic acid (0.14 mmol/ L) were also present to prevent the oxidation of noradrenaline. The PSS was maintained at 37OC and continuously bubbled with a mixture of 95% 0 2 and 5% COZ. After being incubated with [3H]-noradrenaline the artery strips were mounted between two platinum electrodes in an acrylic flow chamber and superfused with PSS at a rate of 2 mL/min. After 30 rnin of superfusion, a 30 s train of 1 ms square wave pulses at a frequency of 2 Hz was delivered to the field electrodes to assist in the clearance of non-specifically bound radioactivity from the tissue. The intrinsic sympathetic nerves of each artery preparation were subjected to field stimulation with two 60 s series of 1 ms monophasic square wave pulses (2 Hz, 20 V). The first of these periods of stimulation was delivered after 120 rnin of superfusion. The interval between the periods of stimulation was 30 min. The effects of tacrine on the resting and stimulation-induced (SI) effluxes of radioactivity were investigated by adding the drug to the PSS superfusing the artery preparations 20 rnin before the second of the two periods of stimulation; the drug then remained present for the duration of the experiment. In some experiments the neuronal uptake-blocking drug desipramine (3 pmol/L) was added to the PSS 10 rnin before the first experimental period of stimulation, and then remained present for the duration of the experiment. In other experiments the monoamine oxidase inhibitor parygline (100 pmol/ L) was added to the PSS 60 rnin before the first period of stimulation and was removed 30 rnin later. The efflux of radioactivity from the pulmonary artery strips was determined from the radioactivity present in 1 min (2 mL) fractions of the PSS that had superfused the,preparations. The resting efflux preceding each of the two experimental periods of stimulation was calculated as the mean content of radioactivity of the three consecutive 1 rnin fractions of superfusate collected immediately before stimulation. The SI efflux of radioactivity for each of the two periods of stimulation was calculated by subtracting the corresponding resting efflux from the content of radioactivity in each of the five consecutive 1 rnin fractions of superfusate collected from the onset of the period of stimulation,
633
and then summing the differences. In each experiment, the resting and SI effluxes for the second period of stimulation were each expressed as a percentage of the corresponding value for the first period of stimulation (% R ~ / Rand I % S2/S1, respectively). This procedure takes account of variation in the amounts of radioactivity released between tissues, and statistical comparison with matching sets of control experiments takes account of changes in resting and SI effluxes due to time alone.
Rat atrial preparations Three series of experiments were performed with rat isolated atrial preparations. For each, albino Wistar rats (250-350 g) of either sex were decapitated and their hearts rapidly removed. The atria were dissected free and placed under a diastolic tension of 9.8 mN between two platinum electrodes in a jacketed organ bath containing 3 mL of PSS. The atrial preparations were allowed to equilibrate for 30 min, during which time the bathing solution was repeatedly changed. In the first series of experiments with atria, the tissues were incubated with [3H]-noradrenaline (3.3 pCi/mL, 0.2 pmol/L) for 20 rnin and were then washed repeatedly with fresh PSS for 60 min. After the first 30 rnin of washing, the atria were subjected to a 30 s period of field stimulation (0.5 ms pulses, 2 Hz) to assist in the removal of loosely-bound radioactive materials. After the washout procedure, the atrial intramural sympathetic nerves were subjected to two periods of field stimulation with series of 20 (0.5 ms) pulses, delivered at a frequency of 1 Hz and a supramaximal voltage of approximately 12 V/cm, given 30 rnin apart. The effects of tacrine on the resting and Sl effluxes of radioactivity were investigated by adding it to the bathing solution 20 rnin before the second of the two experimental periods of stimulation, after which it remained present for the duration of the experiment. The atrial bathing solution was collected at 1 min intervals and the content of radioactivity of each sample determined. The resting and SI induced effluxes were calculated as described above for experiments with pulmonary artery preparations. In some experiments, the neuronal uptake blocking drug 1 pmol/ L desipramine was added to the bathing solution 20 rnin before the first period of stimulation and then remained present throughout. In the second series of experiments with rat atrial preparations, the radioactive efflux from [3H]-noradrenaline-labelled preparations was identified as tritiated noradrenaline and its metabolites. For these experiments, atria from untreated rats and rats treated
634
M.E. Fabiani et al.
with pargyline, as described below, were incubated with ( - )-[ring 2,5,6-3H]-noradrenaline (10 pCi/ mL, 0.24 pmol/L) for 20 min. After 30 rnin of repeated washing with noradrenaline-free PSS 30 pmol/ L tacrine was added to the bathing solution and remained present throughout. In some experiments 1 pmol/L desipramine was introduced to the bathing solution 20 rnin before tacrine and then remained present throughout. The atrial bathing solution was collected during five consecutive 6 min periods commencing 12 rnin before the addition of tacrine. For some experiments the atria were taken from rats pretreated with the monoamine oxidase inhibitor pargyline (100 mg/kg, i.p.) 4 h beforehand. In these experiments pargyline was present in the PSS during the initial 30 min of the equilibration period. The pH of each 6 mL collection of atrial bathing solution was adjusted to 8.6 with 1 mol/L Tris-acetate buffer. A 1 mL aliquot from each fraction was mixed with 10 mL Picofluor (Packard Instruments, IL, USA) and the content of total radioactivity determined. The amounts of tntiated noradrenaline, its tritiated deaminated metabolite 3,4-dihydroxyphenylethylene-glycol (DOPEG) and total non-catechol (O-methylated) metabolites were determined in the remaining amount of each sample of bathing solution collected using a combination of alumina absorption and ion exchange column chromatography, as described by Graefe et al. (1973). Individual tritiated 0-methylated metabolites of [3H]-noradrenaline were not separated but were estimated in terms of the amounts of radioactivity that escaped adsorption during a single passage of the samples (ph 8.6) through alumina columns. Catecholcontaining compounds were eluted from the alumina with acetic acid (0.2 mol/ L), and [3H]-noradrenaline and [jH]-DOPEG in the eluate were separated by passage through columns packed with Dowex 50w resin. The amounts of [3H]-noradrenaline (d/ min) were corrected for recovery, which had a mean value of 86% (s.e.m. f 7). The efficiencies of extraction of the other components of radioactivity were not determined. In the third series of experiments the effects of tacrine on the resting and SI efflux of radioactivity were investigated in atria in which the neuronal transmitter stores of acetycholine had been labelled by incubating the atria with [3H]-choline. The methodology used was essentially that described by Muscholl and Muth (1982) and, subsequently,modified by Loicano and Story (1986). Rat atria were dissected as described above and mounted between two platinum electrodes in a jacketed organ bath containing 3 mL of PSS. Before being incubated with [3H]-choline the atria were incubated with non-radioactive choline chloride
(1 pmol/ L) for 30 min and were subjected to a 5 rnin series of field stimulation with 1 ms pulses at a frequency of 5 Hz and a field strength of 15 V/cm (Lindmar et al, 1980; Muscholl & Muth 1982; Wetzel & Brown 1983). The atria were then incubated with [methyl-3H]- choline chloride (7.8 pCi/ mL, 1.1 .pmol/ L) for 5 rnin and then washed repeatedly for 60 min with PSS containing 10 pmol/ L non-radioactive choline. The non-radioactive choline remained present for the duration of the experiment (Dieterich ez al. 1978). After the first 30 rnin of washing, the atria were subjected to a 30 s period of field stimulation (1 ms pulses, 1 Hz) to assist in the removal of loosely-bound radioactivity. After the washout procedure the intramural cholinergic nerves of the atria were subjected to two periods of field stimulation with series of 30 (1 ms) square wave pulses at a frequency of 1 Hz and a supramaximal voltage of approximately 12 V/cm. The interval between the two periods of stimulation was 30 min. The effects of tacrine on the resting and SI eMuxes of radioactivity were investigated by adding the drug to the bathing solution 20 min before the second period of stimulation. It then remained present for the duration of the experiment. The resting and SI effluxes of radioactivity for each of the two periods of stimulation were determined as for the first series of experiments with [3H]-noradrenaline-labelled atria.
Measurement of radioactivity in samples of
PSS For the measurement of total radioactivity in PSS, each collection of superfusate from pulmonary artery preparations (2 mL) and atrial bathing solution (3 mL) was mixed with 6 and 10 mL, respectively, of Picofluor (PackardInstruments), and the radioactivity present was determined by liquid scintillation counting. Corrections for counting,efficiency were made by external automatic standardization and the results expressed as d/min.
Determination of [3H]-noradrenaline uptake Atria were dissected as described previously and placed into ajacketed organ bath containing 10 mL of fresh PSS. After a 30 min equilibration period the atria were incubated with [3H]-noradrenaline (1 pCi/ mL, 0.07 pmol/L) for6 min. Following incubation the atria were washed with fresh PSS, removed from the organ bath, blotted dry, weighed and then solubilized in glass vials with 2 mL Soluene 350 (Packard Instruments). After solubilization, 10 mL Picofluor was added to each vial, a minimum of 12 h was allowed
635
Effects of tacrine on neuroeffector transmission
for decay of any chemiluminescence and then the radioactivity present was measured by liquid scintillation counting.
periarterial sympathetic nerves and the mean SI efflux evoked by the first period of stimulation (60 s series of pulses at 2 Hz) in control experiments are given in Table 1.
Drugs and radiochemicals
*
The following drugs were used: desipramine hydrochloride (Ciba-Geigy, Australia; Sigma, St Louis, MO, USA); choline chloride (DBH, UK); pargyline hydrochloride (Sigma); tacrine hydrochloride monohydrate (tetrahydroaminoacridine; THA; Institute of Drug Technology, Australia; H. W. Woods, Australia). All drugs were freshly prepared in distilled water. Methyl-[3H]-choline chloride (specific activity 80 Cil mmol) (-){7,8-3H]-noradrenaline (specific activity 10-15 Ci/mmol) and (-)-[ring, 2,5,6-3H]-noradrenaline (specific activity 42.1 Cilmmol) were supplied by the Radiochemical Centre, Amersham, UK.
Statistical analysis of results Data are expressed as means and standard errors of the means (s.e.m.); n represents the number of experiments. The statistical significance of differences between means was determined by testing for differences within groups of means by one-way analyses of variance (ANOVA) followed by planned comparisons (unpaired Student's t-tests, based on pooled variance estimates) between predetermined pairs of means. In all cases probability levels less than 0.05 (P
PREJUNCTIONAL ACTIONS OF TACRINE ON AUTONOMIC NEUROEFFECTOR TRANSMISSION IN RABBIT ISOLATED PULMONARY ARTERY AND RAT ISOLATED ATRIA Maurizio E. Fabiani, Peter Kabo and David F. Story Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia (Received 7 January 1992; revision received 29 May 1992)
SUMMARY 1. This study investigated the effects of tacrine (1,2,3,4-tetrahydr0-9-aminoacridine) on the resting and stimulation-induced (SI) release of radioactive substances from isolated preparations of rat atria and rabbit pulmonary artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline, and from rat atrial preparations in which cholinergic transmitter stores had been labelled with [3H]-acetylcholine. In addition, the effect of tacrine on the uptake of [3H]noradrenaline by noradrenergic nerves in rat atria was determined. 2. Tacrine produced concentration-dependent increases in the resting efflux of radioactivity from both the [3H]-noradrenaline-loaded artery and atrial preparations. Blockade of neuronal amine transport with desipramine reduced the release of radioactivity evoked by tacrine from atria but not that evoked from artery preparations. Inhibition of monoamine oxidase by pargyline pretreatment markedly reduced the tacrine-evoked release of radioactivity in both atrial and artery preparations. 3. The radioactivity released from [3H]-noradrenaline-labelled rat atrial preparations by 30 pmol/ L tacrine consisted entirely of the deaminated metabolite [3H]-DOPEG. The evoked release of [3H]-DOPEG from atria was reduced by approximately 50% by desipramine (1 pmol/L). When atrial monoamine oxidase had been inhibited by pargyline treatment in vivo and in vitro, 30 pmol/ L tacrine evoked the release of [3H]-noradrenaline instead of [3H]-DOPEG. However, the amounts of [3H]-noradrenaline released by tacrine when monoamine oxidase was inhibited were only about 25% of the amounts of [3H]-DOPEG released in untreated atria. 4. Tacrine, in concentrations of I and 10 pmol/L, enhanced the release of radioactivity evoked by field stimulation of [3H]-noradrenaline-loaded rabbit pulmonary artery preparations. This effect was unaltered by desipramine or pretreatment with pargyline. However, in artery preparations pretreated with pargyline, a high concentration of tacrine (100 pmol/ L) markedly reduced SI efflux. In contrast to the findings with artery preparations, tacrine (1 -30 pmol/ L) did not alter SI efflux in rat atrial preparations. 5. It is concluded that tacrine displaces noradrenaline from intraneuronal transmitter stores of sympathetically-innervatedtissues, and that the displaced amine is totally metabolized by monoamine oxidase before leaving the nerve terminals. When deamination of neuronal cytoplasmic noradrenaline is prevented, only a portion of the noradrenaline displaced from storage vesicles passes to the extracellular space. It is likely that the transfer of cytoplasmic noradrenaline out of the terminals is limited by the activity of the amine transport mechanism.
Correspondence: Associate Professor David F. Story, Department of Pharmacology, IJniversityof Melbourne,Parkville, Vic. 3052, Australia.
632
M. E. Fabiani et al. 6. Tacrine, in concentrations of 30 and 100 pmol/L, reduced the uptake radioactivity by rat atria incubated for 5 min periods in [3H]-noradrenaline to approximately 83 and 26%, respectively, of control uptake. Desipramine was much more potent than tacrine in inhibiting [3H]-noradrenaline uptake: 1 pmol/L desipramine reduced the uptake radioactivity to approximately 18% of the control. 7. Tacrine (30 pmol/ L) did not alter the resting efflux of radioactivity from [3H]-acetylcholinelabelled rat atrial preparations, but it reduced the efflux of radioactivity evoked by stimulation of intramural cholinergic nerves. The inhibition of SI efflux in the [3H]-acetylcholine-labelled atria may have been mediated by acetylcholine that had accumulated as a consequence of the anticholinesterase activity of tacrine at cholinergic nerve terminals. Key words: cholinergic transmission, noradrenergic transmission, potassium channel antagonists, rabbit pulmonary artery, rat atria, tacrine.
INTRODUCTION There has been renewed interest in the anticholinesterase drug tacrine ( 1,2,3,4-tetrahydro-9-aminoacridine; THA) following reports of its beneficial effects in Alzheimer's disease (Summers et al. 1986; Becker k Giacobini 1988; Gauthier et al. 1988; Nyback et al. 1988). The anticholinesterase activity of tacrine was first reported by Shaw and Bentley (1953). Subsequently, it was shown to inhibit both acetycholinesterase and pseudocholinesterase (Heilbronn 1961; Ho & Freeman 1965). In view of the cholinergic deficit of central neurones in Alzheimer's disease (Davis & Maloney 1976; Davis & Mohs 1986), it might be assumed that any therapeutic effect of tacrine in Alzheimer's disease is dependent on its anticholinesterase activity. However, other anticholinesterase drugs such as physostigmine (Stern et al. 1987), muscarinic agonists such as pilocarpine (Caine 1980; Bruno et al. 1986) and arecoline (Tariot 1988), and the acetylcholineprecursor choline (Becker & Giacobini 1988) produce no apparent improvement in the disease. Thus, an action of tacrine other than or in addition to the inhibition of cholinesterase enzymes may be involved in any beneficial effect of the drug in Alzheimer's disease. Indeed Summers et al. (1986) suggested that potassium channelblocking activity may play a role in the putative therapeutic effect of tacrine in Alzheimer's disease. Tacrine has structural similarities to the sodium channel-blocking drug 9-aminoacridine (Yeh 1979; Yamamoto & Yeh 1984) and also to the potassium channel-blockingdrug 4-aminopyridine (Soni & Kam 1982; Wesseling & Agoston 1984; Davidson et al. 1988). Moreover, tacrine has been reported to block sodium channels in myelinated axons of the toad Xenopus laevis (Elinder et al. 1989) and in the giant axons of the worm Myxicola (Schauf & Sattin 1987).
In addition, several studies have shown that tacrine blocks potassium channels in a range of excitable cells, including guinea-pig ventricular myocytes (Osterrieder 1987) and atria (Freeman et al. 1988)' rat hippocampal neurones (Rogawski 1987; Halliwell & Grove 1989), and neurones of Xenopus laevis (Elinder et al. 1989), Myxicola (Schauf & Sattin 1987) and of the snail Lyrnnea stagnalis (Drukarch et al. 1987). There is also evidence to suggest that tacrine may have some blocking activity at voltage-dependent Caz+ channels (Elinder et al. 1989). Relatively little is known about the effects of tacrine on neurotransmitter release. In view of the reports of its beneficial effects in Alzheimer's disease, the interaction of tacrine with neurotransmitter systems is worthy of further investigation. The present study was undertaken to investigate the effects of tacrine on noradrenergic and cholinergic autonomic neuroeffector transmission. The effects of tacrine on symphathetic noradrenergic transmission were investigated in isolated preparations of rabbit pulmonary artery and rat atria in which noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. Its effects on cholinergic transmission were investigated in rat atrial prepara#ionsin which the cholinergic transmitter stores had been YhdiolabetIed with [3H+acetylcholine.
METHODS Rabbit pulmonary artery preparation Rabbits of either sex (2-4 kg) were sacrificed a blow to the neck and exsanguinated. The chest was opened and the heart rapidly removed. A segment of the common pulmonary artery was dissected free and cut
Effects of tacrine on neuroeffector transmission
into two spiral strips approximately 20 mm long and 2 mm wide. The artery strips were incubated for 20 rnin with ['HI-noradrenaline (10 pCi/mL, 0.67 pmol/ L), contained in 1 mL of physiological salt solution (PSS) in a jacketed organ bath. The PSS had the following composition (mmol/L): NaCl ( I 18); KCl(4.7); CaC12 (2.5); MgS04 (0.45); NaHC03 (25); KH2P04 (1.03); and D-( +)-glucose (1 I. 1). Disodium edetate (0.067 mmol/ L) and ascorbic acid (0.14 mmol/ L) were also present to prevent the oxidation of noradrenaline. The PSS was maintained at 37OC and continuously bubbled with a mixture of 95% 0 2 and 5% COZ. After being incubated with [3H]-noradrenaline the artery strips were mounted between two platinum electrodes in an acrylic flow chamber and superfused with PSS at a rate of 2 mL/min. After 30 rnin of superfusion, a 30 s train of 1 ms square wave pulses at a frequency of 2 Hz was delivered to the field electrodes to assist in the clearance of non-specifically bound radioactivity from the tissue. The intrinsic sympathetic nerves of each artery preparation were subjected to field stimulation with two 60 s series of 1 ms monophasic square wave pulses (2 Hz, 20 V). The first of these periods of stimulation was delivered after 120 rnin of superfusion. The interval between the periods of stimulation was 30 min. The effects of tacrine on the resting and stimulation-induced (SI) effluxes of radioactivity were investigated by adding the drug to the PSS superfusing the artery preparations 20 rnin before the second of the two periods of stimulation; the drug then remained present for the duration of the experiment. In some experiments the neuronal uptake-blocking drug desipramine (3 pmol/L) was added to the PSS 10 rnin before the first experimental period of stimulation, and then remained present for the duration of the experiment. In other experiments the monoamine oxidase inhibitor parygline (100 pmol/ L) was added to the PSS 60 rnin before the first period of stimulation and was removed 30 rnin later. The efflux of radioactivity from the pulmonary artery strips was determined from the radioactivity present in 1 min (2 mL) fractions of the PSS that had superfused the,preparations. The resting efflux preceding each of the two experimental periods of stimulation was calculated as the mean content of radioactivity of the three consecutive 1 rnin fractions of superfusate collected immediately before stimulation. The SI efflux of radioactivity for each of the two periods of stimulation was calculated by subtracting the corresponding resting efflux from the content of radioactivity in each of the five consecutive 1 rnin fractions of superfusate collected from the onset of the period of stimulation,
633
and then summing the differences. In each experiment, the resting and SI effluxes for the second period of stimulation were each expressed as a percentage of the corresponding value for the first period of stimulation (% R ~ / Rand I % S2/S1, respectively). This procedure takes account of variation in the amounts of radioactivity released between tissues, and statistical comparison with matching sets of control experiments takes account of changes in resting and SI effluxes due to time alone.
Rat atrial preparations Three series of experiments were performed with rat isolated atrial preparations. For each, albino Wistar rats (250-350 g) of either sex were decapitated and their hearts rapidly removed. The atria were dissected free and placed under a diastolic tension of 9.8 mN between two platinum electrodes in a jacketed organ bath containing 3 mL of PSS. The atrial preparations were allowed to equilibrate for 30 min, during which time the bathing solution was repeatedly changed. In the first series of experiments with atria, the tissues were incubated with [3H]-noradrenaline (3.3 pCi/mL, 0.2 pmol/L) for 20 rnin and were then washed repeatedly with fresh PSS for 60 min. After the first 30 rnin of washing, the atria were subjected to a 30 s period of field stimulation (0.5 ms pulses, 2 Hz) to assist in the removal of loosely-bound radioactive materials. After the washout procedure, the atrial intramural sympathetic nerves were subjected to two periods of field stimulation with series of 20 (0.5 ms) pulses, delivered at a frequency of 1 Hz and a supramaximal voltage of approximately 12 V/cm, given 30 rnin apart. The effects of tacrine on the resting and Sl effluxes of radioactivity were investigated by adding it to the bathing solution 20 rnin before the second of the two experimental periods of stimulation, after which it remained present for the duration of the experiment. The atrial bathing solution was collected at 1 min intervals and the content of radioactivity of each sample determined. The resting and SI induced effluxes were calculated as described above for experiments with pulmonary artery preparations. In some experiments, the neuronal uptake blocking drug 1 pmol/ L desipramine was added to the bathing solution 20 rnin before the first period of stimulation and then remained present throughout. In the second series of experiments with rat atrial preparations, the radioactive efflux from [3H]-noradrenaline-labelled preparations was identified as tritiated noradrenaline and its metabolites. For these experiments, atria from untreated rats and rats treated
634
M.E. Fabiani et al.
with pargyline, as described below, were incubated with ( - )-[ring 2,5,6-3H]-noradrenaline (10 pCi/ mL, 0.24 pmol/L) for 20 min. After 30 rnin of repeated washing with noradrenaline-free PSS 30 pmol/ L tacrine was added to the bathing solution and remained present throughout. In some experiments 1 pmol/L desipramine was introduced to the bathing solution 20 rnin before tacrine and then remained present throughout. The atrial bathing solution was collected during five consecutive 6 min periods commencing 12 rnin before the addition of tacrine. For some experiments the atria were taken from rats pretreated with the monoamine oxidase inhibitor pargyline (100 mg/kg, i.p.) 4 h beforehand. In these experiments pargyline was present in the PSS during the initial 30 min of the equilibration period. The pH of each 6 mL collection of atrial bathing solution was adjusted to 8.6 with 1 mol/L Tris-acetate buffer. A 1 mL aliquot from each fraction was mixed with 10 mL Picofluor (Packard Instruments, IL, USA) and the content of total radioactivity determined. The amounts of tntiated noradrenaline, its tritiated deaminated metabolite 3,4-dihydroxyphenylethylene-glycol (DOPEG) and total non-catechol (O-methylated) metabolites were determined in the remaining amount of each sample of bathing solution collected using a combination of alumina absorption and ion exchange column chromatography, as described by Graefe et al. (1973). Individual tritiated 0-methylated metabolites of [3H]-noradrenaline were not separated but were estimated in terms of the amounts of radioactivity that escaped adsorption during a single passage of the samples (ph 8.6) through alumina columns. Catecholcontaining compounds were eluted from the alumina with acetic acid (0.2 mol/ L), and [3H]-noradrenaline and [jH]-DOPEG in the eluate were separated by passage through columns packed with Dowex 50w resin. The amounts of [3H]-noradrenaline (d/ min) were corrected for recovery, which had a mean value of 86% (s.e.m. f 7). The efficiencies of extraction of the other components of radioactivity were not determined. In the third series of experiments the effects of tacrine on the resting and SI efflux of radioactivity were investigated in atria in which the neuronal transmitter stores of acetycholine had been labelled by incubating the atria with [3H]-choline. The methodology used was essentially that described by Muscholl and Muth (1982) and, subsequently,modified by Loicano and Story (1986). Rat atria were dissected as described above and mounted between two platinum electrodes in a jacketed organ bath containing 3 mL of PSS. Before being incubated with [3H]-choline the atria were incubated with non-radioactive choline chloride
(1 pmol/ L) for 30 min and were subjected to a 5 rnin series of field stimulation with 1 ms pulses at a frequency of 5 Hz and a field strength of 15 V/cm (Lindmar et al, 1980; Muscholl & Muth 1982; Wetzel & Brown 1983). The atria were then incubated with [methyl-3H]- choline chloride (7.8 pCi/ mL, 1.1 .pmol/ L) for 5 rnin and then washed repeatedly for 60 min with PSS containing 10 pmol/ L non-radioactive choline. The non-radioactive choline remained present for the duration of the experiment (Dieterich ez al. 1978). After the first 30 rnin of washing, the atria were subjected to a 30 s period of field stimulation (1 ms pulses, 1 Hz) to assist in the removal of loosely-bound radioactivity. After the washout procedure the intramural cholinergic nerves of the atria were subjected to two periods of field stimulation with series of 30 (1 ms) square wave pulses at a frequency of 1 Hz and a supramaximal voltage of approximately 12 V/cm. The interval between the two periods of stimulation was 30 min. The effects of tacrine on the resting and SI eMuxes of radioactivity were investigated by adding the drug to the bathing solution 20 min before the second period of stimulation. It then remained present for the duration of the experiment. The resting and SI effluxes of radioactivity for each of the two periods of stimulation were determined as for the first series of experiments with [3H]-noradrenaline-labelled atria.
Measurement of radioactivity in samples of
PSS For the measurement of total radioactivity in PSS, each collection of superfusate from pulmonary artery preparations (2 mL) and atrial bathing solution (3 mL) was mixed with 6 and 10 mL, respectively, of Picofluor (PackardInstruments), and the radioactivity present was determined by liquid scintillation counting. Corrections for counting,efficiency were made by external automatic standardization and the results expressed as d/min.
Determination of [3H]-noradrenaline uptake Atria were dissected as described previously and placed into ajacketed organ bath containing 10 mL of fresh PSS. After a 30 min equilibration period the atria were incubated with [3H]-noradrenaline (1 pCi/ mL, 0.07 pmol/L) for6 min. Following incubation the atria were washed with fresh PSS, removed from the organ bath, blotted dry, weighed and then solubilized in glass vials with 2 mL Soluene 350 (Packard Instruments). After solubilization, 10 mL Picofluor was added to each vial, a minimum of 12 h was allowed
635
Effects of tacrine on neuroeffector transmission
for decay of any chemiluminescence and then the radioactivity present was measured by liquid scintillation counting.
periarterial sympathetic nerves and the mean SI efflux evoked by the first period of stimulation (60 s series of pulses at 2 Hz) in control experiments are given in Table 1.
Drugs and radiochemicals
*
The following drugs were used: desipramine hydrochloride (Ciba-Geigy, Australia; Sigma, St Louis, MO, USA); choline chloride (DBH, UK); pargyline hydrochloride (Sigma); tacrine hydrochloride monohydrate (tetrahydroaminoacridine; THA; Institute of Drug Technology, Australia; H. W. Woods, Australia). All drugs were freshly prepared in distilled water. Methyl-[3H]-choline chloride (specific activity 80 Cil mmol) (-){7,8-3H]-noradrenaline (specific activity 10-15 Ci/mmol) and (-)-[ring, 2,5,6-3H]-noradrenaline (specific activity 42.1 Cilmmol) were supplied by the Radiochemical Centre, Amersham, UK.
Statistical analysis of results Data are expressed as means and standard errors of the means (s.e.m.); n represents the number of experiments. The statistical significance of differences between means was determined by testing for differences within groups of means by one-way analyses of variance (ANOVA) followed by planned comparisons (unpaired Student's t-tests, based on pooled variance estimates) between predetermined pairs of means. In all cases probability levels less than 0.05 (P
