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Notes: Summary The resting efflux of choline from perfused chicken hearts varied from 0.4 to 2.6 nmol/g min, but was constant for at least 80 min in the individual experiments. The rate of choline efflux was found to be equal to the rate of choline formation in the heart, which, from the following reasons, was essentially due to hydrolysis of choline phospholipids. (1) Cardiac content of choline phospholipids (7,200 nmol/g) was much higher than that of acetylcholine (5.5 nmol/g). (2) Resting release of acetylcholine was 0.016 nmol/g min and, after inhibition of cholinesterase, only about 0.1 nmol/g min. Resting efflux of choline was reduced by mepacrine, a phospholipase A2 inhibitor, by perfusion with a Ca2+-free Tyrode's solution containing EGTA and by the combination mepacrine plus Ca2+-free/EGTA solution. In all experiments the reduced choline efflux levelled off within 10 min at about 50%. Omission or elevation of Mg2+ from 1.05 to 10.5 mmol/l had no effect. Resting efflux was increased to 150% by oleic acid (as sodium salt; 2×10−5 mol/l) which is known to activate phospholipase D. Likewise muscarinic agonists (carbachol and acetylcholine) caused facilitation of the efflux of endogenous choline that was blocked by 3×10−7 mol/l atropine. This effect was not reduced, but even slightly enhanced, by mepacrine and by infusion of EGTA in a modified Tyrode's solution (Ca2+-free, 10.5 mmol/l Mg2+). It is concluded that the resting efflux of choline from the heart is essentially due to hydrolysis of choline phospholipids, that half of the efflux is insensitive to mepacrine and is Ca2+-independent (excluding an involvement of phospholipase A2). Moreover, this Ca2+-independent efflux is facilitated by muscarinic agonists, possibly through an effect on phospholipase D activity.

Notes: Summary The resting efflux of choline into the perfusate (Tyrode's solution) of isolated hearts was equal to the rate, at which choline was liberated from phospholipid degradation (Lindmar et al. 1986). 1. Infusion of isoprenaline (2×10−7 mol/l), forskolin (1–3×10−6 mol/l) or 3-isobutyl-1-methylxanthine (IBMX; 3×10−4 mol/l) for 40 min markedly enhanced the efflux of choline. The increase was linear during the experimental period and, in the case of isoprenaline, was blocked by 3×10−7 mol/l atenolol. 2. In the guinea-pig heart, IBMX at a threshold concentration of 10−4 mol/l shifted the concentration-response curve for the effect of forskolin on the efflux of choline to the left by one log unit. 3. Forskolin (10−6 mol/l) increased also the tissue content of cyclic AMP. This effect and the increase of choline efflux evoked by forskolin were blocked by 2×10−7 mol/l carbachol. Likewise, inhibition of cholinesterase activity caused by diisopropylfluorophosphate antagonized the forskolin-evoked acceleration of choline efflux indicating a response to endogenous acetylcholine. The muscarinic inhibition of the enhanced choline efflux was reversed by 3×10−7 mol/l atropine. 4. The phospholipase A2 inhibitor mepacrine as well as infusion of a low Ca2+-Tyrode's solution (0.2 instead of 1.8 mmol/l) blocked the effect of forskolin on choline efflux, whereas the generation of cyclic AMP by forskolin was unaffected by low Ca2+-solution. It is concluded that, in the heart, the degradation of phospholipids as reflected by the release of free choline was stimulated by β-adrenoceptor activation, an effect which was mediated by generation of cyclic AMP. The enzyme of the phospholipid degradation pathways that was activated (directly or indirectly) by cyclic AMP seems to be an internal Ca2+-dependent phospholipase A2 or C.

Notes: Summary 1. The concentrations of acetylcholine, choline and noradrenaline were estimated in the perfusate (overflow) of isolated hearts of chickens, cats, rabbits and guinea pigs. Neurotransmitter release was evoked by stimulation of both vagus nerves and by direct stimulation of the heart (field stimulation). 2. In the absence of exogenous choline and physostigmine, field stimulation at 20 Hz for 20 min caused an overflow of acetylcholine from the hearts of the 4 species investigated. During vagal stimulation, however, acetylcholine was detected only in the perfusate of the chicken heart. 3. Field stimulation for 2 min caused an overflow of 193 pmol g−1 min−1 acetylcholine and of 666 pmol g−1 min−1 noradrenaline from the guinea pig heart; pretreatment of the animals with reserpine blocked the release of noradrenaline but left the overflow of acetylcholine unaltered. 4. When the overflow of acetylcholine was evoked by vagal stimulation, infusion of 10−5 M choline into the cat and chicken heart caused an increase in the overflow that was 2–3-fold in the chicken heart and at least 23-fold in the cat heart (23 times the limit of estimation). In the presence of choline, the overflow of acetylcholine from the hearts of the 4 species evoked by field stimulation was 2–3 times the overflow in the absence of the drug. 5. Inhibition of the cholinesterase activity by 10−6 M physostigmine raised the overflow of acetylcholine evoked by vagal and/or by field stimulation uniformly by a factor of 2 to 3 in the 4 species investigated. In the cat heart, the combination of 10−5 M choline and 10−6 M physostigmine increased the overflow evoked by field stimulation for 20 min from 0.54 to 3.6 nmol g−1 20 min−1, i.e. by a factor of 7. 6. The cardiac content of acetylcholine was highest in the chicken heart (9.8 nmol/g) and lowest in the guinea pig heart (2.1 nmol/g). 7. The spontaneous efflux of choline from the isolated hearts after 15 min of perfusion ranged from 0.4 (cat) to 2.1 nmol g−1 min−1 (chicken) and was maintained at these levels for at least 1 h. 8. In the blood of chickens, cats and rabbits, the choline concentration was 0.5–1.0×10−5 M. 9. It is concluded that (1) an appreciable amount of acetylcholine released from parasympathetic nerves escapes into the circulation of isolated hearts, (2) the extent of the extracellular hydrolysis of acetylcholine is the same in avian and mammalian hearts, (3) the release of acetylcholine evoked by vagal stimulation is much smaller in the isolated cat heart than that in the chicken heart, because of an insufficient ganglionic transmission due to a deficiency in extracellular choline and finally (4) the amount of acetylcholine released by vagal stimulation is dependent on both the efflux of choline from extraneuronal sources and the overall density of the cholinergic innervation of the heart.

Notes: Summary 1. Isolated rabbit hearts were perfused with (−)-noradrenaline, (−)-adrenaline and (±)-isoprenaline for various time periods (1–180 min) and then washed with an amine-free medium. The venous concentration of the amine was estimated fluorimetrically during the infusion and after its end, to study removal and efflux, respectively. 2. In untreated hearts and after pretreatment with reserpine the removal had a constant rate over 20–60 min. After pretreatment with pargyline to block monoamine oxidase (MAO), however, the removal of noradrenaline declined exponentially to zero. Inhibition of the neuronal uptake (desipramine) and chemical sympathectomy (6-hydroxydopamine) abolished the removal of noradrenaline. Isoprenaline was not removed to any significant extent. 3. The efflux of noradrenaline originated in 4 different compartments as indicated by the various efflux components. The half-times in untreated hearts were about 0.1, 0.4 and 7 min, and after block of intraneuronal inactivation (pargyline plus reserpine) 0.1, 0.4 and 43 min. 4. The 1st compartment (t 1/2=0.1 min) represents an open compartment including the vascular space. Several results indicate that the 2nd compartment is identical with the extracellular space. Inhibition of amine uptake by desipramine caused a strong increase of the total output resulting from the 2nd phase of efflux which then contributed a fraction of 0.21 to the total initial rate of efflux. Furthermore, endogenous noradrenaline released by sympathetic nerve stimulation and by DMPP was washed out of the heart into the perfusate with nearly the same half-time (t 1/2=0.32 min) as that found for the 2nd phase of efflux following noradrenaline infusion (t 1/2=0.41 min). The 3rd compartment of untreated hearts (t 1/2=7 min) is not identical with that found after block of both MAO and vesicular uptake (t 1/2=43 min). The former compartment was smaller and more rapidly equilibrated, and access of catecholamines was not depressed by inhibition of neuronal uptake or by chemical sympathectomy. On the other hand, the compartment occurring after block of intraneuronal inactivation became smaller—or even disappeared—after inhibition of neuronal uptake or after chemical sympathectomy; this latter compartment was not affected by block of vesicular uptake. It is concluded that the 3rd compartment of untreated hearts is located extraneuronally; an intraneuronal compartment could not be detected in these hearts under efflux conditions. The 4th noradrenaline compartment, occuring as 3rd phase of efflux after block of MAO, is located within the adrenergic nerves but outside the vesicles; therefore this compartment is identical with, or is part of, the axoplasm. 5. The efflux of isoprenaline originated in 3 different compartments of which the 1st and 2nd (t 1/2=0.1 and 0.3 min) seem to be identical with the corresponding noradrenaline compartments. The 3rd isoprenaline compartment must be extraneuronal since the slow isoprenaline efflux, even after block of MAO, was similar to that of noradrenaline from untreated hearts. In contrast, after block of intraneuronal inactivation the slow adrenaline efflux was identical with the neuronal efflux of noradrenaline. 6. Since the half-time of elimination of noradrenaline from the axoplasmic compartment increased with increasing initial rates of efflux (Y o), it was assumed that a capacity-limited process was involved in the neuronal efflux. This can be exhaustion of intraneuronal binding sites, saturation of efflux or enzymatic degradation.

Notes: Summary The efflux of acetylcholine, of radioactively labelled acetylcholine and choline, into the venous effluent of the perfused chicken heart was studied to determine the kinetics of both interstitial washout and hydrolysis of acetylcholine. Stimulation of both cervical vagus nerves (e.g., for 5 s at 40 Hz) caused a release of acetylcholine, which appeared partially unhydrolyzed in the venous effluent, and reduced force of contraction and heart rate. For comparison labelled acetylcholine or choline was infused for 5 s into the heart and again the venous efflux of either substance was determined. It was found that the kinetics of efflux of acetylcholine or choline from the interstitial space were of first order. The mean half times were 16.2 s (after infusion of acetylcholine) and 17.9 s (after nerve stimulation) for acetylcholine and 17.9 s (after infusion of choline) for choline. In the interstitial space, radioactivity (sum of [3H]-acetylcholine and [3H]-choline formed from [3H]-acetylcholine) released by nerve stimulation declined mono-exponentially with a rate constant of 0.069 s−1 and a half time of 10 s (due to washout), whereas the concentration of unhydrolyzed [3H]-acetylcholine decreased in a multi-exponential fashion due to both washout and hydrolysis. The interstitial concentration of [3H]-acetylcholine reached the 50% level after 2.5 s. In conclusion, the long persistence of unhydrolyzed acetylcholine in the interstitial space of the heart appears to be due to an apparently low rate of hydrolysis. This, in turn, is responsible for the importance of diffusion and washout of acetylcholine from the interstitial space as significant factors of synaptic removal of acetylcholine. Moreover, the results support the notion that the sustained interstitial concentration of acetylcholine determines the long duration of cardiac responses to vagal stimulation.