Search

Low renin hypertension probably does not represent a clinical entity. In many patients with low renin hypertension blood pressure is normalized by treatment with diuretics only; in these patients a (genetic?) sensitivity to salt might play a predominant role in the pathogenesis of hypertension and renin suppression. In another group of patients renin suppression appears to be secondary to the hypertensive process. This is indicated by the observation that prevalence of low renin hypertension increases with age and that it is more frequent in advanced stages of hypertension. Also a diminished sympathetic tone might play a part in the renin unresponsiveness. Finally, although no positive evidence was found, the possibility cannot be excluded that, at least in some cases, a mineralocorticoid other than aldosterone is involved. Neither in normotensive subjects nor in hypertensive patients, both with normal and with low plasma renin was a correlation between plasma renin concentration and plasma aldosterone concentration following stimulation by upright posture found. More detailed studies will be necessary to clarify the relationship between the renin-angiotensin system and aldosterone secretion during upright posture, in particular in patients with low renin hypertension.

Tacrine, 10(-6) M, enhanced the acetylcholine (ACh) overflow evoked in perfused chicken hearts by field stimulation (5 Hz, 1 min) from 183 to 346 pmol g-1 min-1. Increase to the same level were observed after pretreatment with diisopropylfluorophosphate (DFP) as well as after DFP plus 10(-6) M tacrine. Tacrine, 10(-5) M, caused further enhancement with or without DFP up to 851 pmol g-1 min-1. It was concluded that 10(-6) M tacrine enhanced the ACh overflow by choline esterase inhibition, whereas 10(-5) M tacrine caused, in addition, an increase of neuronal ACh release.

The efflux of choline was determined in rat striatal slices, incubated chicken atria and perfused chicken hearts. 4 beta-Phorbol-12 beta,13 alpha-dibutyrate (PDB) and 4 beta-phorbol-12 beta-myristate, 13 alpha-acetate (PMA) were used to stimulate protein kinase C. The other phorbol esters, 4 beta-phorbol-13 alpha-acetate (PAc) and 4 alpha-phorbol-12 beta,13 alpha-didecanoate (4 alpha PDD), known to be inactive, were tested to evaluate the specificity of the responses. PDB markedly enhanced the efflux of choline in all of the three preparations. The PDB-evoked efflux of choline in incubated chicken atria was equal to the net production of choline and, therefore, was not caused by translocation of intracellular free choline. After inhibition of the cholinesterase activity, PDB linearly increased the efflux of choline in rat striatal slices, but failed to alter the spontaneous efflux of acetylcholine. Thus acetylcholine did not serve as the source of the PDB-evoked efflux of choline. PMA was as effective as PDB, whereas PAc and 4 alpha PDD failed to alter the choline efflux in the perfused heart. Both infusion of a Ca2(+)-free EGTA-containing Tyrode solution and mepacrine reduced the spontaneous efflux of choline by about 40% and blocked the PDB-evoked efflux of choline. In contrast, a Ca2(+)-free solution without EGTA failed to alter the spontaneous and the PDB-evoked choline efflux. It is concluded that phorbol esters stimulate the hydrolysis of choline-containing phospholipids in heart and brain via activation of protein kinase C.

Activation of phospholipase D (PLD) and phosphoinositide-specific phospholipase C (PI-PLC) by fluoride, to stimulate heterotrimeric G-proteins, and by phorbol esters, to stimulate protein kinase C (PKC), was studied in rat atria. Fluoride and 4beta-phorbol-12beta,13alpha-dibutyrate (PDB), in contrast to 4beta-phorbol-13alpha-acetate (PAc), activated PLD, catalyzing the formation of [3H]-phosphatidylethanol ([3H]-PETH), [3H]-phosphatidic acid ([3H]-PA), choline and sn-1,2-diacylglycerol (DAG). Basal PLD activity was resistant to drastic changes in Ca2+ and to Ro 31-8220, a PKC inhibitor, but was decreased by genistein, an inhibitor of tyrosine kinase, and increased by vanadate, a tyrosine phosphatase inhibitor; both effects were, however, very small. Fluoride-evoked PLD activity was resistant to Ro 31-8220 and to genistein, but was Ca2+-dependent. The rate of fluoride-induced PLD activation was maintained for at least 60 min. In contrast, PDB-mediated PLD activity was blocked by Ro 31-8220 and was resistant to extracellular Ca2+-depletion and desensitized within ca. 15 min. PDB markedly potentiated the fluoride-evoked generation of [3H]-phosphatidylethanol and of choline, but inhibited the formation of [3H]-inositol phosphates ([3H]-IP(1-3)). Ethanol (2%) blocked the PDB-evoked generation of both [3H]-phosphatidic acid and of sn-1,2-diacylglycerol, whereas fluoride-evoked responses were reduced only to approximately 50%. In conclusion, the trimeric G-protein-PLD pathway in heart tissue did not enclose PKC activation and was long-lasting and Ca2+-dependent; there was no evidence for an involvement of tyrosine phosphorylation. However, PKC activation modulated G-protein-coupled PLD and PI-PLC activities in opposite directions. PLD activity significantly contributed to the mass production of sn-1,2-diacylglycerol in the heart. The evidence for a pathophysiological role of PLD activation in cardiac hypertrophy and in ischemic preconditioning is discussed.

Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF inf4 sup− ; 10 mM NaF plus 10 μM AlCl3). Atria of rats and chickens were incubated with [3H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [3H]-phosphatidyl-ethanol (PETH) was rapidly formed at the expense of [3H]-PtdOH. This transphosphatidylation reaction is specific for phospholipase D activity. The basal formation of PETH was not inhibited by a Ca2+-free, EGTA-containing medium. - The phorbol ester 4β-phorbol-12β,13α-dibutyrate (PDB), which is known to activate protein kinase C, enhanced the net formation of choline, whereas the inactive 4β-phorbol-13α-acetate (PAc) was ineffective. PDB (0.2 μM), in contrast to PAc, also increased the formation of [3H]-PtdOH and, in the presence of ethanol, of [3H]-PETH. The PDB-evoked formation of PETH occurred again at the expense of PtdOH. Treshold and maximum effective concentrations of PDB were 10 nM and 0.2–0.6 μM, respectively. The effects of PDB on either choline efflux and generation of PETH showed the same Cat+-dependency, i.e., both effects were blocked by a Ca2+-free, EGTA-containing medium, but not by a Ca2+-free medium without EGTA. In protein kinase C-deficient tissue which was prepared by pretreatment with 0.61 μM PDB for 27 h, PDB failed to enhance the formation of PtdOH and PETH. - A1F4−, a known activator of G-proteins, increased not only the tissue content of inositol phosphates, but also markedly enhanced choline efflux and formation of [3H]-PtdOH and PETH. In conclusion, in mammalian and avian atria a high phospholipase D activity was found even under basal conditions. The enzyme was stimulated by protein kinase C and presumably by a G protein.

The 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.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine and thereby seems to play a key role in a novel pathway of signal transduction. PLD activity in rat hippocampal slices and atria of rat, guinea pig and chicken hearts was determined by measuring the catalytic products choline (Ch), phosphatidic acid (PA) and, in the presence of a primary alcohol, phosphatidylpropanol or phosphatidylethanol. It was found that the PLD activity was high, even under resting conditions, in both tissues, especially in the hippocampus, and that the enzyme activity could be enhanced by activation of protein kinase C and by muscarinic receptor stimulation.

1. Electrically driven chicken and guinea-pig atria were used to investigate the negative inotropic effects of the muscarinic agonists methacholine and acetylcholine (ACh). The release of ACh from isolated hearts into the perfusate in response to (preganglionic) vagal or (pre- and postganglionic) field stimulation was bioassayed on the guinea-pig ileum or determined by labelling with [3H]-choline. 2. Concentration-response curves for the negative inotropic effect of methacholine were shifted to the right by pirenzepine in various concentrations (0.03 to 10 mumol l-1). The pA2 values were 7.76 in chicken atria and 6.53 in guinea-pig atria. Pirenzepine and atropine antagonized the negative inotropic response to 0.3 mumol l-1 ACh. The half-maximally effective concentrations (IC50) of pirenzepine (Pz) and atropine were 40 and 5.4 nmol l-1 in chicken atria and 330 and 3.5 nmol l-1, respectively, in guinea-pig atria. Thus, the respective potency ratios (IC50Pz/IC50atropine) were 7.4 and 94.3 in the two species. 3. Pirenzepine in low concentrations increased the release of unlabelled and 3H-labelled ACh from isolated hearts evoked by vagal and field stimulation only in chicken, but not in guinea-pigs. The half-maximally-effective concentration of pirenzepine was about 30 nmol l-1 in the chicken heart, whereas, in the guinea-pig heart, an increased release was observed at 300 nmol l-1. 4. (+)-Tubocurarine [(+)-Tc; 100 mumol l-1] reduced the release of ACh evoked by (preganglionic) vagal stimulation to a (+)-Tc-resistant release of about 30%. The time-course of the neuronal release of [3H]-ACh was markedly altered: the onset was delayed and the termination was extended beyond the period of stimulation (1 min or 5s) by several seconds. The (+)-Tc-resistant release was nearly abolished by 30 nmol l-1 pirenzepine. 5. In conclusion, the pre- and post-synaptic muscarinic receptors of the parasympathetic neuroeffector junction of the heart both belong to the M1-subtype in the chicken and to an M2-subtype in the guinea-pig. Block of the nicotinic ganglionic transmission in the chicken heart by (+)-Tc unmasked a muscarinic transmission, which presumably was mediated through M1-receptors stimulating a low and prolonged postganglionic release of ACh.

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 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.

Corpuscles of Stannius (CS) from the salmonid Salmo irideus L. were shown to contain considerable amounts of noradrenaline (NA), adrenaline (A) and 5-hydroxytryptamine (5-HT). Fluorescence microscopy (Falck-Hillarp technique) revealed the presence of intensely fluorescent cells and green fluorescent “adrenergic” nerve fibres running in the proximity of blood vessels and endocrine cells (EC). The intensely fluorescent cells resembled chromaffin cells and exhibited granular vesicles of the NA-type. They received cholinergic synapses with typical pre- and postsynaptic membrane specializations. Adrenergic nerve fibres contained small and large granular vesicles and a small proportion of agranular vesicles. They came within 120 nm of EC becoming devoid of their satellite cell sheath. The presence of nerve fibres and the amount and distribution of biogenic amines in CS suggest that a functional link with the EC moiety cannot be excluded. It may be that NA, A and 5-HT are stored within the same nerve fibre.

Stimulation of the cervical vagus nerves caused an output of acetylcholine (ACh) from the isolated chicken heart, which almost exclusively was released from the postganglionic neurons: (+)-tubocurarine (3 X 10(-14) M) reduced the output to 12 +/- 6% (n = 7) of the control. Stimulation of the two nerve trunks ws equally effective in releasing ACh.--Evidence that a large number of postganglionic neurons receives bilateral innervation was based on two experimental series. (1). The sum of the ACh outputs evoked by unilateral (separate) nerve stimulation of the right and the left vagus was higher than the bilaterally evoked output (100%) and increased with increasing frequencies (10, 20 and 40 Hz) from 115 +/- 13% to 131 +/- 9% (n = 13). In the presence of 10(-4) M 4-aminopyridine, unilaterally evoked output (40 Hz) was further increased from 131 to 176 +/- 5% (n = 21).--(2.) In the presence of 4-aminopyridine plus hemicholinium-3 (2 X 10(-5) M), unilateral nerve stimulation at 40 Hz evoked an output of ACh that decreased from 477 to 79 pmol g-1 min-1 during a 20 min-period of stimulation due to transmitter depletion. Thereafter output of ACh evoked by stimulation of the contralateral nerve was reduced by 73% as compared to the control value (475 pmol g-1 min-1; output without the preceding 20 min-stimulation).--It is concluded that a large number of parasympathetic postganglionic neurons of the chicken heart receives a dual excitatory input from both right and left vagus nerve.

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 (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.

Rabbit 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.

The experiments were carried out in order to investigate the functional significance of substitution of noradrenaline by amines known to exert actions weaker than those of the natural transmitter. On the isolated rabbit heart the potency relative to noradrenaline of (±)-dihydroxyephedrine to increase the rate was 1/4 and the potency to augment the contractile amplitude was 1/6. The corresponding values for (±)-dihydroxypseudoephedrine were 1/67 and 1/140, respectively.

Isolated rabbit hearts were perfused at 34° (control), 24° or 12°C. The neuronal efflux of noradrenaline after perfusion with the amine for 1 h was depressed at 24° C (Q 10 about 5) in the presence or absence of desipramine; at 12°C the efflux was below the limit of estimation. Moderate reduction of the temperature (24° C) decreased the removal of perfused noradrenaline to about 60% of the control value and caused a 1.7-fold increase of the output of noradrenaline evoked by sympathetic nerve stimulation. It is concluded that the extremely temperature-dependent efflux of noradrenaline across the axonal membrane is not part of the release of noradrenaline evoked by nerve stimulation.

Threo-corbadrine caused a long-lasting depletion of noradrenaline in the heart and in mesenteric vessels and lowered the blood pressure of normal and renal hypertensive rats. It is suggested that threo-cobadrine decreases vascular tone by acting peripherally as a substitute adrenergic transmitter.

1. The effect of infusion of a modified Tyrode's solution (“high K+-low Na+ solution”) into the isolated chicken heart on the content of acetylcholine in the tissue and the overflow of acetylcholine were compared to those evoked by vagal stimulation. 2. The release of acetylcholine was measured over 15-min periods of either stimulation of the vagus nerves (40 V, 1 ms) at 20 Hz or of infusion of the high K+-low Na+ solution (108 mM K+, 44 mM Na+). 3. Both stimuli caused a maximum overflow of acetylcholine in the first few minutes whether or not 10−6 M physostigmine was present. The overflow was maintained during the vagal stimulation at a constant rate of at least 35% the initial rate, whereas the overflow evoked by the high K+-low Na+ solution ceased after 5 min. Under the latter conditions, the size of the acetylcholine store available for release (overflow in the presence of physostigmine) was 41% of the total store. 4. Physostigmine (10−6 M) caused a 2–3-fold increase of the overflow of acetylcholine evoked either by nerve stimulation or by infusion of the high K+-low Na+ solution. Reduction of [Ca2+]o from 1.8 to 0.2 mM strongly reduced the overflow caused by either kind of stimulation. 5. The sum of the overflow evoked by the high K+-low Na+ solution and the cardiac content of acetylcholine after the stimulation did not exceed the cardiac content before stimulation. 6. In conclusion, infusion of a high K+-low Na+ solution caused an overflow of acetylcholine in the absence and presence of physostigmine that was similar to that evoked by nerve stimulation with respect to Ca2+-dependency and hydrolysis of the released acetylcholine. The rate of synthesis of acetylcholine maintaining a steady state release was high during nerve stimulation, but was blocked during infusion of the high K+-low Na+ solution, presumably by block of the high affinity uptake of choline.

Low renin hypertension probably does not represent a clinical entity. In many patients with low renin hypertension blood pressure is normalized by treatment with diuretics only; in these patients a (genetic?) sensitivity to salt might play a predominant role in the pathogenesis of hypertension and renin suppression. In another group of patients renin suppression appears to be secondary to the hypertensive process. This is indicated by the observation that prevalence of low renin hypertension increases with age and that it is more frequent in advanced stages of hypertension. Also a diminished sympathetic tone might play a part in the renin unresponsiveness. Finally, although no positive evidence was found, the possibility cannot be excluded that, at least in some cases, a mineralocorticoid other than aldosterone is involved. Neither in normotensive subjects nor in hypertensive patients, both with normal and with low plasma renin was a correlation between plasma renin concentration and plasma aldosterone concentration following stimulation by upright posture found. More detailed studies will be necessary to clarify the relationship between the renin-angiotensin system and aldosterone secretion during upright posture, in particular in patients with low renin hypertension.

DMPP (1,1-dimethyl-4-phenylpiperazine) in various concentrations between 1.6×10−6 M and 6.2×10−5 M was infused into isolated rabbit hearts to study the neuronal release and uptake of noradrenaline.

Publisher Summary This chapter discusses a study to analyze the effects of aminopyridines on parasympathetic neuro-effector transmission in the heart. The release of acetylcholine (ACh) was determined in isolated, perfused chicken hearts. In the absence of aminopyridines (APs), the stimulation of both vagus nerves increased the output of ACh into the perfusate from 24 to 280 pmol/g min, when an anticholinesterase agent was not present. 4-Aminopyridine (4-AP) or 3,4-diaminopyridine (3,4-DAP) enhanced the evoked release of ACh. When, under control conditions, the ACh release was maximal, the APs were ineffective or much less effective (less than 2-fold increase) than at a low rate of release (up to 10-fold increase). The potency of 3,4-DAP was about 10-fold higher than that of 4-AP. As was expected from the AP-evoked facilitation of ACh release, the two compounds markedly increased the negative and chronotropic effects of vagal stimulation predominantly at low frequencies in isolated atria of various species. The compounds did not alter force and rate of contraction at concentrations that maximally facilitated transmitter release.

Untersuchungen der letzten Jahre haben gezeigt, das die Reninsekretion vom sympathischen Nervensystem beeinflust wird [3]. Weiterhin ist bekannt, das 20 bis 30% aller Patienten mit essentieller Hypertonie ein erniedrigtes Plasmarenin aufweisen [1]. Es stellte sich die Frage, ob Patienten mit essentieller Hypertonie und normalem Plasmarenin (NRH) sich im Funktionszustand des Sympathikus von Patienten mit niedrigem Plasmarenin (LRH) unterscheiden.

The 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.

1 The concentrations of noradrenaline and adrenaline in various organs, arterial plasma and venous outflow from isolated hearts of adult chickens have been determined. 2 The relative adrenaline concentrations (percentage of the sum of noradrenaline and adrenaline) in the heart (33%), spleen (16%) and brain (26%) were higher than those found in mammalian organs. Chemical sympathectomy by pretreatment with 6-hydroxydopamine caused a decrease of the noradrenaline and adrenaline concentrations in the heart to 20 and 23% and in the spleen to 16 and 29%, respectively. 3 Stimulation of the right sympathetic nerves, infusion of tyramine or infusion of a modified Tyrode solution containing 108mM K+ and 44 mM Na+ caused an output of both noradrenaline and adrenaline into the perfusate of isolated hearts. The relative adrenaline concentration in the perfusate (20-28%) was not significantly different from the relative adrenaline concentration remaining in these hearts (19-22%). In the individual experiments, the noradrenaline: adrenaline ratios of the stimulation perfusates were positively correlated with the ratios found in the hearts. 4 The effects of noradrenaline and adrenaline on cardiac rate and tension development were studied in spontaneously beating right atria and electrically driven left atria, respectively. In addition, the arterial pressure rise in response to noradrenaline or adrenaline was;measured in chickens. It was found that the cardio-vaseart rate, cardiac tension development and arterial blood pressure, was not significantly different from that of adrenaline. 5 It is concluded that, in the chicken heart and spleen, both noradrenaline and adrenaline act as sympathetic neutrotransmitters.

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 mumol/L) was lower than that of the sinus blood. The resulting cerebral arterio-venous difference of choline was negative (3.2 mumol/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 choline level 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor activation modulates functions of the heart and neurotransmission in the peripheral and central nervous system. Moreover, muscarinic agonists produce changes in the metabolism of, for example, heart tissue, such as inhibition of beta-adrenoceptor-mediated cAMP accumulation, glycogenolysis and lipase activation.

Medullary cells were enzymatically dissociated from adult bovine adrenal medullae by a perfusion technique using collagenase and maintained in culture for up to 4 weeks. The perfusion technique was a simplified modification of that described by Livett & Co-Workers ( Fenwick, Fajdiga, Howe & Livett , 1978 ) and included cyclical perfusions of cortex-free medullae via the central vein at 37 C for up to 90 min with three 30 ml batches of 0.5% collagenase-containing phosphate buffered saline or Hanks's balanced salt solution and a final trituration step of the minced tissue. The cells were cultured on collagen-coated glass coverslips in modified Rose chambers, on collagen-coated Petri dishes or in Falcon flasks, studied by phase contrast and electron microscopy, catecholamine cytochemistry and assayed for adrenaline and noradrenaline. When grown on collagen about 50% of the catecholamine-storing cells extended short, broad processes as judged by catecholamine cytochemistry using the glyoxylic acid method. These processes rarely grew longer than 60 μm and many of them were seen to retract after 1 week in culture. Process formation was more pronounced and increased with time, when cells were grown on plastic surfaces. Fifteen to 20% of these cells formed long varicose axons after 18 days in culture. Fibre outgrowth from bovine chromaffin cells was also observed, when pieces of medullary tissue were transplanted on to the sympathetically denervated iris in the anterior eye chamber of Nunu mice. Addition of either nerve growth factor (10–1000 ng/ml 2.5 S nerve growth factor) or antibodies to nerve growth factor (1.5 μg/ml) did not affect fibre outgrowth in vitro . Ultrastructural examinations revealed that the vast majority of cells in our cultures were typical chromaffin cells. Using the different densities of primary and secondary amine storing granular vesicles as a criterion for distinguishing noradrenaline- and adrenaline-containing cells we found a significant decrease in the number of adrenaline-containing cells with time. By 3 weeks virtually all chromaffin cells contained storage vesicles typical of noradrenaline. Chromaffin storage vesicles appeared to be more densely packed and the amount of rough endoplasmic reticulum was increased in cells treated with nerve growth factor. Results obtained by biochemical analyses indicated that chromaffin cells lost about 50% of their original amine content and stored equal amounts of noradrenaline and adrenaline from day 9 onwards compared to an initial noradrenaline/adrenaline ratio of approximately 1:6. Administration of nerve growth factor did not significantly alter the proportion and levels of adrenaline and noradrenaline in cultured bovine chromaffin cells. This investigation reveals that the capacity of bovine catecholamine-storing cells to form neurite-like processes differs considerably from that which corresponding cells from young rat adrenal medullae exhibit in vitro ( Unsicker, Krisch, Otten & Thoenen , 1978 b ). Chromaffin cells from adult bovine adrenal medullae kept in culture retain a large number of features typical of differentiated cells over a considerable length of time. Thus they may be profitably used to study long-term effects of drugs and interactions with other cells at morphological and biochemical levels.

The occurrence of unhydrolyzed acetylcholine (ACh) in the cardiac perfusate during vagal stimulation in the absence of cholinesterase inhibition has been demonstrated by several methods. Because some ACh was found unhydrolyzed in the extracellular space for several seconds after vagal stimulation (half-time of decay 2.5 s), it appears that the prolonged time course of the cardiac responses to bursts of vagal activity is determined by a slow rate of transmitter inactivation (diffusion plus hydrolysis) in addition to slowly operating postsynaptic mechanisms mediated by activation of the muscarinic receptor. The neuronal uptake of choline in isolated heart preparations was found to be Na+ dependent, sensitive to hemicholinium 3, and activated by vagal stimulation. Activation occurred after a delay of 1 or 2 min and slowly faded within 5 min after stimulation. Resting release of ACh was insensitive to extracellular Ca2+ and to muscarinic feedback inhibition, in contrast to the evoked transmitter release. Inasmuch as atropine increased ACh release by vagal and field stimulation to the same extent, muscarinic feedback inhibition is likely to occur at postganglionic parasympathetic neurons. Adrenergic agonists and propranolol did not significantly change the release of ACh.

In the heart, choline phospholipids were by far the largest fraction (about 50%) of phospho-lipids, much larger than that of inositol phospholipids (less than 6%) and phosphatidic acid (0.3%). The choline phospholipids (11 μmol/g) maintained a constant efflux of choline of about 1.5 nmol g −1 min −1 into the perfusate. Carbachol (10 μm) rapidly enhanced the choline efflux by a muscarinic mechanism, that was independent of mepacrine, an inhibitor of phospholipase A 2 , as well as of extracellular Ca 2+ the maximum acceleration was reached within 2 min. In contrast, the accumulation of inositol phosphates by carbachol was blocked in the presence of a Ca 2+ -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 Ca 2+ -free, EGTA-containing solution. Sodium oleate (20 μM), 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.

Effects of (-)-N-ethylephedrine (etafedrine) were studied on the tracheal chain preparation and the atria of the guinea pig and on the rabbit perfused heart. Contraction of the tracheal chain by acetylcholine or histamine was antagonized by epinephrine (adrenaline), etafedrine and ephedrine, the relative potencies being 91:1:0.3, respectively. Propranolol (5 X 10(-7) mol/l) completely antagonized the bronchodilator effect of 10(-4) mol/l etafedrine on the acetylcholine-evoked contraction. Etafedrine up to 3 X 10(-4) mol/l did not increase heart rate or force of contraction in guinea pig atria. In contrast to tyramine and ephedrine (both 10(-5) mol/l), etafedrine (10(-4) mol/l) failed to release 3H-norepinephrine (noradrenaline) in the perfused rabbit heart. Moreover, the concentration-dependent positive chronotropic and inotropic effects of norepinephrine in isolated atria were slightly, but not significantly enhanced by etafedrine. It is concluded that N-ethylation of ephedrine suppresses the indirect sympathomimetic activity and markedly enhances the efficacy on beta 2-adrenoceptors. Etafedrine appears to be a sympathomimetic agent with a selective activity on beta 2-adrenoceptors.

The effects of a single high dose (10mg/kg) of vinblastine (vb) sulfate (“Velbe”, Lilly) on the ultrastructure, catecholamine (CA) content and activity of CA-synthesizing enzymes of the rat adrenal medulla were studied for up to 120h after intravenous injection of the drug.

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). Infusion of isoprenaline (2 X 10(-7) mol/l), forskolin (1-3 X 10(-6) mol/l) or 3-isobutyl-1-methylxanthine (IBMX; 3 X 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 X 10(-7) mol/l atenolol. 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. 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 X 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 X 10(-7) mol/l atropine. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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. Cardiac content of choline phospholipids (7,200 nmol/g) was much higher than that of acetylcholine (5.5 nmol/g). 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 X 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 X 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).(ABSTRACT TRUNCATED AT 250 WORDS)

Vier normotone Versuchspersonen erhielten an 3 aufeinanderfolgenden Tagen je 1, 0 g α-Methyldopa (α-MDopa) in 4 Einzeldosen zu 0, 25 g alle 6 Std, die letzte Dosis um 24 Uhr am 3. Tag. Zwei Harnportionen wurden am 3. Behandlungstag sowie an den beiden darauffolgenden Tagen gesammelt, jeweils eine Ruheperiode (Bettruhe) von 4–7 Uhr und eine Aktivitatsperiode (normale Labortatigkeit) von 7–11 Uhr. Die Bestimmung von –-MDopa, –-Methyldopamin (–-MDA), Noradrenalin (NA), Adrenalin (A) und –-Methylnoradrenalin (–-MNA) im Harn erfolgte wie fruher angegeben (Muscholl u. Rahn [1]).

After oral administration of α-methyldopa to six volunteers α-methylnoradrenaline was excreted in the urine. Sympathetic reflex responses induced by physical activity caused a parallel increase in the excretion of noradrenaline and α-methylnoradrenaline. This confirms the hypothesis that α-methylnoradrenaline is released as an adrenergic ‘false’ transmitter in man.