R. Lindmar

Die Wirkung von Pharmaka auf die Elimination von Noradrenalin aus der Perfusionsflssigkeit und die Noradrenalinaufnahme in das isolierte Herz

In den letzten Jahren wurde in einer Reihe yon Arbeiten aus verschiedenen Laboratorien gezeigt, dab in die Zirkulation gelangtes Noradrenalin in sympathisch innervierte Gewebe aufgenommen wird. Definiert und experimentell bestimmt wurde diese Aufnahme entweder als Zunahme der Noradrenalinkonzentration des Gewebes gegeniiber der Konzentration des endogenen Noradrenalins nach Verabreichung yon L-Noradrenalin (MvscHOLL 1960, 1961; STROMBLAD U. NICKERSO~; CAMrOS u. Sm-DV~MAN) oder als Konzentration von tt3-Noradrenalin im Gewebe nach Verabreichung von D,L-HS-Noradrenalin (Wm~rBY, AXELROD U. WEIL-MALHERBE; HERTTING, AXELROD U. WttITBY). In einigen Versuchen mit H3-Noradrenalin wurden auch spezifische Aktivit~ten von Noradrenalin im Gewebe mitgeteilt (HV, RTTI~G U. Hv, SS; POTTS, R U. A X ~ O D ) . IVV,~SS, N mal~ am perfundierten Rattenherzen die spezifische Aktivit~t des au/genommenen Noradrenalins und erfaBte dadurch die GrSl3e des Austausches yon H3-Noradrenalin mit dem endogenen Noradrenalin. Danach dfirfte bei den Konzentrationsmessungen yon H3-Noradrenalin in Geweben in den Versuchen der Arbeitsgruppe yon AXELROD im wesentlichen eine Nettoaufnahme erfal~t worden sein. Eine zweite Methode zur Bestimmung der Noradrenalinaufnahme besteht darin, die arteriovenSse Differenz der Noradrenalinkonzentration bei einem yon Blur oder einer PerfusionslSsung durchstrSmten Organ zu messen (SI]sGs.L, GILMORV, U. SA~NOrF; Ln~DMA~ U. MUSCHOLL 1962;

Unterschiede zwischen Tyramin und Dimethylphenylpiperazin in der Ca++-Abhängigkeit und im zeitlichen Verlauf der Noradrenalin-Freisetzung am isolierten Kaninchenherzen

On the perfused rabbit heart a constant infusion of tyramine released noradrenaline continuously and independently of the external Ca++ concentration. In contrast, noradrenaline release by DMPP was only transient and required the presence of Ca++.

Neuronal and extraneuronal uptake and efflux of catecholamines in the isolated rabbit heart

Summary1.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 (t1/2=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 (t1/2=0.32 min) as that found for the 2nd phase of efflux following noradrenaline infusion (t1/2=0.41 min). The 3rd compartment of untreated hearts (t1/2=7 min) is not identical with that found after block of both MAO and vesicular uptake (t1/2=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 (t1/2=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 (Yo), 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.

On the mechanism of muscarinic hydrolysis of choline phospholipids in the heart

In the heart, choline phospholipids were by far the largest fraction (about 50%) of phospholipids, much larger than that of inositol phospholipids (less than 6%) and phosphatidic acid (0.3%). The choline phospholipids (11 mumol/g) maintained a constant efflux of choline of about 1.5 nmol g-1 min-1 into the perfusate. Carbachol (10 microM) rapidly enhanced the choline efflux by a muscarinic mechanism, that was independent of mepacrine, an inhibitor of phospholipase A2, as well as of extracellular Ca2+; the maximum acceleration was reached within 2 min. In contrast, the accumulation of inositol phosphates by carbachol was blocked in the presence of a Ca2+-free perfusion medium. Similar to the carbachol-evoked choline efflux, the increase in tissue content of phosphatidic acid by carbachol was unaffected by infusion of a Ca2+-free, EGTA-containing solution. Sodium oleate (20 microM), an activator of phospholipase D, imitated the effects of carbachol on choline and phosphatidic acid, whereas NaF (5 mM), which has been reported to inhibit phospholipase D, blocked carbachol-evoked efflux of choline. In conclusion, muscarinic receptor stimulation enhanced the hydrolysis of choline phospholipids presumably via activation of phospholipase D. The immediate formation of choline, phosphatidic acid and presumably diacylglycerol is discussed including its possible physiological importance.

The role of choline in the release of acetylcholine in isolated hearts

Summary1.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.

Characterization of choline efflux from the perfused heart at rest and after muscarine receptor activation

SummaryThe 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 Tyrodes 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 Tyrodes 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.

The neuronal efflux of noradrenaline: Dependency on sodium and facilitation by ouabain

SummaryRabbit hearts were isolated after pretreatment with the MAO inhibitor pargyline and with reserpine and were perfused with 200 ng/ml noradrenaline for 1 h. During the subsequent wash-out with an amine-free solution for 2 h, the neuronal efflux of noradrenaline declined mono-exponentially with a mean halftime of 42 min. Both Na+-free solution and ouabain caused facilitation of the efflux which thereafter declined in a multi-exponential fashion. The maximum facilitation was reached after 3 min of Na+-free perfusion and 25 min after introduction of ouabain. The amount of exogenous noradrenaline accumulated in the heart was only partially released when the extracellular Na+-concentration was normal, whereas perfusion with low Na+-solution evoked a complete release of the accumulated noradrenaline. In conclusion, the noradrenaline leaving the adrenergic neurone with the neuronal efflux originates preferentially in one intraneuronal compartment, when the external Na+-concentration is normal. Reduction of external Na+-concentration, however, seems to evoke the release from multiple intraneuronal compartments in series. The effect of Na+-depletion can be explained by the Na+-gradient hypothesis, but evidence is still lacking. The normal Na+-gradient seems to serve as a prerequisite for the axoplasmic retention of noradrenaline under the present conditions.

Ouabain enhances release of acetylcholine in the heart evoked by unilateral vagal stimulation

SummaryThe aim of the study was to elucidate peripheral effects of ouabain on the parasympathetic innervation of the heart, effects that could contribute to the experimentally and clinically well established “vagal effect of cardiac glycosides”. The experiments were carried out with ouabain concentrations of 3×10−7 and 10−6 mol/l, which were considered “therapeutic”, as they increased force of contraction and did not elicit arrhythmias in incubated chicken atria.In atrial preparations of chickens and guinea-pigs the negative chronotropic and inotropic effects of acetylcholine (ACh) were not altered by 3×10−7 mol/l ouabain. Resting efflux of ACh from perfused chicken hearts was increased by ouabain from 10 to a maximum of 30 pmol/g min, whereas release of ACh evoked by bilateral vagal stimulation at 3 or 20 Hz for 1 min was unchanged (resting release subtracted). In contrast, release of ACh caused by unilateral vagal stimulation was augmented by ouabain up to 200% of the control. Release by unilateral stimulation (80 pmol/g; 20 Hz) was calculated for each experiment by averaging the releases evoked by consecutive stimulation of the right and left nerves. Ouabain infused for 90 min did not alter the tissue content of ACh (5.5 nmol/g).Within 2 days after unilateral (left) vagal transsection (denervation of cardiac ganglia) the release of ACh evoked by stimulation of the intact nerve (20 Hz) increased from about 80 to 200 pmol/g, whereas the release from the lesioned nerve markedly declined. One day after denervation, ouabain had lost the ability to facilitate the release of ACh evoked by stimulation of the intact nerve, whereas the release by stimulation of the lesioned nerve was still increased.It is concluded that ouabain at “therapeutic” concentrations increased resting release of ACh but did not influence the mechanism of action potential-evoked release of ACh. The effect of exogenous ACh on sinus node activity was not enhanced by ouabain. The observation that ouabain increased release of ACh caused by unilateral, but not by bilateral vagal stimulation was explained by an increase in the number of activated postganglionic neurons arising from those (contralateral) ganglia that received a subthreshold input from the stimulated vagus nerve.

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart

SummaryThe effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [3H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (±)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenidol > AF-DX 116. The comparison with known pA2 values for M1-, M2- and M3-receptors revealed that the presynaptic autoreceptor meets the criteria of an M1-receptor. Basal, not electrically evoked overflow of unlabelled ACh into the perfusate was caused by ‘leakage’ release (non-exocytotic), as it was independent of extracellular Ca2+ . Muscarinic receptor antagonists failed to enhance basel overflow. In contrast, when ChE activity was inhibited by 10−6M tacrine or pretreatment with 10−4M DFP, the ACh overflow was partially Ca2+-dependent and was reduced by tetrodotoxine. Moreover, block of the inhibitory muscarinic autoreceptors by (±)-telenzepine or pirenzepine caused a several-fold enhancement of the ACh release. The potencies of these antagonists were identical to those found for the electrically evoked [3H]-ACh release. The rate of ACh release enhanced by ChE inhibition plus telenzepine corresponds to about 12% of the total ACh pool per min, which is about the maximum amount of ACh that is available for any kind of stimuli. The release was dependent on the presence of exogenous choline. Hence elevation of ACh release led to a correspondingly enhanced ACh synthesis. The dramatic enhancement of ACh release by the ChE inhibition in combination with a block of presynaptic muscarinic autoinhibition was not inhibited by (+)-tubocurarine but by atropine (10−9 to 10−7 M) or 10−6 M telenzepine. It is concluded that basal release of ACh in the heart was due to non-exocytotic ‘leakage’ release. Inhibition of ChE led to a marked stimulation of excitatory muscarinic receptors of the intrinsic parasympathetic neuron with a consecutive postganglionic release of ACh. The strong postganglionic excitation was obvious when the inhibitory muscarinic autoreceptors were selectively blocked. Of the two described muscarinic receptors found in the parasympathetic postganglionic neuron of the chicken heart only the inhibitory was classified as being M1, whereas the subtype of the excitatory one is unlike M1 and remains to be identified.

Muscarinic mobilization of choline in rat brain in vivo as shown by the cerebral arterio-venous difference of choline.

Abstract: In anesthetized rats, the choline levels of cerebrospinal fluid and plasma obtained from blood collected from peripheral vessels (carotid artery, cardiac vessels) and from the transverse sinus were determined with a radioenzymatic assay. Cortical release of choline was studied using the “cup technique.” The plasma choline level of the peripheral blood (11.5 μmol/L) was lower than that of the sinus blood. The resulting cerebral arterio‐venous difference of choline was negative (3.2 μmol/L) and reflected the net release of choline from the whole brain. The plasma choline levels were not different irrespective of whether the rats were anesthetized with ether, urethane, or pentobarbital. However, the choline level of the cerebrospinal fluid, which normally was lower than the plasma choline levels, was increased by urethane anesthesia to a level between the arterial and venous plasma concentrations of the brain. In old rats (24 months), the choline level of the cerebrospinal fluid was significantly lowered, when compared with the results obtained with younger rats (2–4 months). In rats kept on a low‐choline diet for 2 weeks, the plasma choline level of the peripheral blood was reduced to 51% of the control. The effect on the choline level of the sinus blood was smaller; the cerebral arterio‐venous difference of choline was not reduced (it was even slightly enhanced). Likewise, the cholinelevel of the cerebrospinal fluid and the cortical release of choline were not altered. Intraperitoneal administration of oxotremorine in pentobarbital‐anesthetized rats kept on a low‐choline diet increased the plasma levels of choline. The rise of the plasma level of the peripheral blood was blocked by atropine and by methylatropine. Atropine alone or in combination with oxotremorine reduced the arterio‐venous difference to half the level obtained by oxotremorine or oxotremorine plus methylatropine. In conclusion, the net formation of choline in the whole brain is partially controlled by central cholinergic activity and is unaltered even at a considerably reduced supply of choline from the peripheral circulation. Moreover, the remarkable maintenance of a certain choline level of the cerebrospinal fluid at a reduced (low‐choline diet) or elevated (oxotremorine) plasma level also suggests that the extracellular concentration of choline in the brain is held at a constant level eventually at the expense of cellular phospholipids.