Effects of cocaine and desipramine on the neurally evoked overflow of endogenous noradrenaline from the rat heart.

1 Stimulation of postganglionic cardiac sympathetic nerves produced a stimulation frequency-dependent overflow of endogenous noradrenaline from the otherwise isolated rat heart. 2 Such nerve stimulation also produced increases in heart rate. There was significant correlation between heart rate increases and corresponding noradrenaline concentrations in the coronary venous effluent. 3 Cocaine (3 X 10(-5)M) caused a significant reduction in both the noradrenaline overflow and the heart rate increase, produced by nerve stimulation for 1 min at 4 Hz. 4 Desipramine (10(-6)M) caused a significant increase in the noradrenaline overflow produced by stimulation for 1 min (4 Hz) with a mean increase of approximately 60%. There was no significant effect on the heart rate increase produced by such stimulation. 5 The opposite effects of cocaine (3 X 10(-5)M) and desipramine (10(-6)M) on noradrenaline overflow are attributed to differences in the local anaesthetic properties of these agents.     

Effect of blockade of noradrenaline re-uptake on evoked tritium overflow from mouse vasa deferentia and rat cortex slices.

1. In tissues previously incubated with [3H]-noradrenaline exposure to cocaine (0.1 to 10 microM) or desmethylimipramine (0.01 to 1 microM) produced a concentration-dependent increase (up to 2 fold) in electrically evoked (3 Hz, 2 ms, 20 mA, 120s every 20 min) fractional overflow of tritium from rat brain cortex slices but not from mouse vas deferens (2.5 Hz, 2 ms, 400 mA, for 90s every 14 min). 2. Yohimbine and idazoxan (0.01 to 1 microM) increased fractional evoked overflow of tritium by up to 2 fold; in the presence of these drugs, cocaine (10 microM) produced an increase in both tissues (up to 3.5 fold over control). 3. In brain slice an increase in stimulation frequency (0.1, 0.5, 1, 3 and 6 Hz) decreased fractional evoked overflow of tritium per pulse but cocaine (10 microM) produced a significant enhancement at each frequency except 6 Hz. In vas deferens fractional tritium overflow per pulse changed little with increasing frequency and cocaine produced no effect. 4. In both tissues fractional evoked overflow of tritium was dependent on the stimulation current; cocaine (10 microM) increased fractional evoked overflow from brain slice at every current tested but was without effect in vas deferens. 5. Chromatographic separation of the released tritium showed there was little difference in the proportions of [3H]-noradrenaline and 3H-metabolites overflowing from the tissues. Cocaine increased the proportion of [3H]-noradrenaline and decreased the proportion of [3H]-DOPEG overflowing both at rest and during stimulation. 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of endogenous noradrenaline release from intact and epithelium-denuded rat isolated trachea.

1. Overflow of endogenous noradrenaline (NA) from the in vitro incubated rat trachea evoked by two periods of electrical field stimulation (S1, S2 at 3 or 15 Hz) or by high potassium (60 mM) was determined by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. In the presence of the neuronal uptake inhibitor desipramine, the alpha 2-adrenoceptor antagonist, yohimbine, enhanced the overflow of NA evoked by stimulation at 3 Hz by about 100% suggesting the presence of presynaptic inhibitory autoreceptors on the sympathetic nerves innervating the trachea. 3. When desipramine and yohimbine were present throughout the experiments, the overflow of NA evoked by the second period of electrical stimulation (S2) was significantly smaller than that evoked by the first (S1). This decline of overflow was prevented when the NA precursor, tyrosine, was additionally present throughout the experiments. 4. After removal of the epithelium, the tissue content of NA was reduced by about 30%, suggesting that part of the NA may be present and released within the epithelium. However, the overflow of NA evoked by stimulation at 3 Hz or 15 Hz was reduced by 70-80%, indicating that the epithelium may additionally exert a permissive role on the release of NA within the airways, possibly by suppressing inhibitory factors. 5. Stimulation by high potassium (60 mM for 10 min) caused a large overflow of NA (about 45% of the tissue NA), both from epithelium-free and epithelium-denuded tracheae. Thus the 'endogenous inhibition' of NA release after removal of the epithelium is surmountable when a high potassium stimulus is applied.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of aminophylline on the acutely transplanted dog heart: effect of alpha- and beta-adrenoceptor blockade.

1 Aminophylline inhibits the coronary vasodilator actions of adenosine. Our previous studies suggested that low dose infusions of aminophylline reduce coronary blood flow in the isolated heart. In the present study we investigated the actions of aminophylline on coronary blood flow and myocardial contractility in a transplanted heart model. Drugs were given by close coronary arterial infusion. 2 Aminophylline in low doses (200 mug/min) reduced coronary blood flow by 21 plus or minus 2% (mean plus or minus s.e. mean) but did not alter myocardial contractility or heart rate. Higher doses (500 and 1000 mug/min) increased coronary blood flow and myocardial contractility without changing heart rate. 3 Alpha-adrenoceptor blockade with phenoxybenzamine did not affect the response to a low dose of aminophylline (200 mug/min). 4 Propranolol in doses of 10 and 30mug/min blocked beta-adrenoceptors but did not change coronary blood flow. The higher dose reduced myocardial contractility. 5 The effects of a high dose of aminophylline (1000 mug/min) on coronary blood flow were not changed by either alpha- or beta-adrenoceptor blockade, although propranolol (30 mug/min) reduced the augmentation in myocardial contractility. 6 The results show that when given in doses which do not alter myocardial contractility, aminophylline reduces coronary blood flow in the isolated heart and that this is not mediated through an alpha-adrenoceptor mechanism. They also show that the increases in coronary blood flow and positive inotropic effects obtained with higher doses of aminophylline are not mediated through catecholamines and suggest that higher doses of aminophylline have a small direct coronary vasodilator action. The low dose vasoconstrictor response may be produced by inhibition of the coronary vasodilator action of locally produced adenosine.     

Effects of human recombinant interleukins on stimulation-evoked noradrenaline overflow from the rat perfused spleen

Experiments were carried out in the isolated spleen of the rat to study in a lymphoid organ the influence of interleukins (ILs) on noradrenaline release. Spleens were perfused with Tyrode's solution and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection.Perivascularelectrical stimulation (4 or 10 Hz, 20–28 mA, 2 min) caused an increase in noradrenaline overflow and in perfusion pressure, both of which were markedly reduced by perfusion with Ca²⁺-free solution, abolished by tetrodotoxin, unaffected by hexamethonium, and subject to ₂-adrenoceptor- and muscarinic receptor-mediated modulation as shown by the effects of rauwolscine and methacholine. Human recombinant IL-1ß and IL-2 and mouse recombinant IL-2 10ng/ml failed to affect the evoked overflow of noradrenaline after an exposure time of 15 min. In contrast, human recombinant IL-1ß and IL-2 0.1 ng/ml reduced the evoked overflow after exposure for 80 min; the inhibition tended to increase 30 min later despite washout. Murine recombinant IL-2 1.2 ng/ml caused no change after contact with the tissue for 80 min but there was an inhibition 30 min later after washout. Human recombinant IL-6 (0.1 ng/ml) caused no significant change.The inhibitory effect of low concentrations of IL-1ß and IL-2 supports the idea that locally produced mediators of the immune system may affect neuronal function. Correspondence to: I. T. Bognar at the above address     

In vitro studies of endogenous noradrenaline and NPY overflow from the rat hypothalamus during maturation and ageing

Marked changes in brain monoamine content and NPY content occur during maturation and ageing. Earlier in vivo studies in our laboratory have reported blunted K⁺ stimulated noradrenaline release and reduced NPY overflow in aged animals using microdialysis and push pull techniques. In this study, in vitro superfusion techniques were established to measure endogenous noradrenaline, NPY, DOPAC and 5-HIAA overflow from the hypothalamus of 1, 5 and 16 month old Sprague-Dawley rats. A period of high K⁺ (56 mM) stimulation was carried out to elicit maximal release. Basal noradrenaline overflow was similar in all age groups of rats and during K⁺-induced depolarisation similar 3–4 fold increases were observed. On the other hand, basal and K⁺ stimulated NPY overflow were significantly greater in the adult rats compared to 1 month and 16 month old rats. Despite differences in absolute NPY overflow, the relative increase over resting was not significantly different across age groups. The molar quantities of hypothalamic NPY overflow at rest and under K⁺ stimulated conditions were three orders of magnitude lower than noradrenaline. Results of these studies suggest that both NPY and noradrenaline can be released from a similar hypothalamic pool. Basal and K⁺-evoked DOPAC and 5-HIAA overflow were similar between the 3 age groups. Thus the overflow of hypothalamic noradrenaline, DOPAC and 5-HIAA under in vitro conditions was not altered from 1 to 16 months. In contrast, 5 month old rats had significantly higher NPY overflow than the other age groups (P < 0.05), consistent with a reported decline in NPY content with advanced age. Hypothalamic noradrenaline overflow was not affected by ageing, suggesting that a selective loss of NPY in the arcuo-PVN projection, or other projections to the hypothalamus with ageing may contribute to the reduction in NPY overflow in aged rats. Received: 9 June 1997 / Accepted: 6 November 1997     

Reduction by beta-adrenoceptor blockade of hypoxia-induced right heart hypertrophy in the rat.

1. The study was undertaken to assess the role of beta-adrenoceptors in the induction of compensatory cardiac hypertrophy in an in vivo model. 2. In the rat, exposure to severe hypoxia (6% inspired oxygen for 8 h day) caused a 51% increase in right heart weight and a 75% increase in haematocrit. 3. The hypoxia-induced right ventricular hypertrophic response was reduced by 65% by oral treatment with a high dose of the non-selective beta-adrenoceptor antagonist, propranolol (80 mg kg-1 body weight); the drug treatment caused only a minor reduction (6%) in secondary polycythaemia. 4. With a less severe degree of hypoxia (7% inspired oxygen) there was only minimal secondary polycythaemia (+15%), and a lesser degree of compensatory right ventricular hypertrophy in untreated rats (+33%). 5. Treatment with the beta 1-adrenoceptor antagonist, atenolol, in a dose of 80 mg kg-1 body weight abolished right ventricular hypertrophy in response to 7% inspired oxygen, without affecting haematocrit and caused a small reduction in the ratio of heart weight to body weight in normoxic rats. 6. The results show that the effect of propranolol on hypoxic right ventricular hypertrophy is not secondary to any effect on secondary polycythaemia as has previously been suggested and that a marked reduction of compensatory cardiac hypertrophy can be obtained by a beta 1-selective adrenoceptor antagonist. Thus these findings support the view that noradrenaline released from cardiac sympathetic nerve terminals exerts a trophic effect on myocardial cells and demonstrates that in vivo, this trophic effect can be reduced by beta 1-adrenoceptor blockade.     

The uptake and overflow of radiolabelled beta-adrenoceptor blocking agents by the isolated vas deferens of the rat.

1. A comparison of uptake into and overflow from the isolated vas deferens of the rat has been made between [3H]-noradrenaline ([3H]-NA), [14C]-D-sorbitol and three radio-labelled beta-adrenoceptor blocking agents, [14C]-practolol, [14C]-(+/-)-propranolol and [3H]-penbutolol. 2. The accumulation of [3H]-NA after 30 min incubation was reduced by desmethylimipramine (DMI) 1 X 10(-8)M and was also reduced in vasa from rats pretreated with 6-hydroxydopamine (6-OHDA). This was not so with [14C]-D-sorbitol. 3. 6-OHDA pretreatment of the rats reduced the uptake of [3H]-penbutolol after 30 min incubation but not that of [4C]-propranolol or [14C]-practolol. DMI 1 X 10(-8)M did not alter the tissue uptake of [14C]-propranolol, [14C]-practolol or [3H]-penbutolol. 4. Electrical stimulation of vasa preloaded with [3H]-NA caused a significantly greater increase in [3H]-NA overflow than during the resting, unstimulated periods. No such increase in overflow was observed with [14C]-sorbitol or any of the three beta-adrenoceptor blocking agents use. 5. The beta-adrenoceptor blocking agent penbutolol was shown to possess adrenergic neurone blocking activity in the isolated vas deferens of the rat. 6. It is concluded that any effect that practolol or (+/-)-propranolol have on noradrenergic neurones is brought about without the need for these drugs to gain access to the interior of the neurone.     

Effects of noradrenaline on rat paratracheal neurones and localization of an endogenous source of noradrenaline.

1. Intracellular recording techniques were used to study the actions of exogenous noradrenaline (NA) on rat paratracheal neurones in situ. The receptor subtypes underlying these actions were investigated by application of selective adrenoceptor antagonists. 2. Application of NA (0.1-10 microM) by superfusion evoked a membrane depolarization in 85% (52 out of 61) of all paratracheal neurones studied. The response consisted of a slow depolarization which was sometimes accompanied by action potential discharge. In 26 out of 31 cells the response was associated with a change in input resistance of the cell membrane. In 22 out of 26 cells there was a 30% increase, whilst in a further 4 cells there was a 15% decrease in input resistance. The amplitude of the NA depolarization was concentration-dependent. 3. The depolarization evoked by NA was reversibly antagonized by prazosin (1 microM) but unaffected by yohimbine (1 microM) or propranolol (1-10 microM). 4. High performance liquid chromatography with electrochemical detection (h.p.l.c.-e.c.d.) was used to assay for NA and dopamine in samples containing mainly paratracheal ganglia and in samples of tracheal smooth muscle with mucosa. NA was present in all samples assayed at a level of 1.6 micrograms NA g-1 and 0.5 microgram NA g-1 wet weight of the two sample types respectively. Dopamine was not detected in any samples of either ganglia or smooth muscle with mucosa. 5. It is concluded that NA-evoked depolarizations of rat paratracheal neurones result from stimulation of alpha 1-adrenoceptors, and that local levels of NA may be sufficiently high to activate these receptors directly.     

Prejunctional histamine H3-receptors inhibit electrically evoked endogenous noradrenaline overflow in the portal vein of freely moving rats

The effects of intra-arterial injection of different doses of the selective histamine H₃-receptor agonist R-α-methylhistamine and the selective histamine H₃-receptor antagonist thioperamide on basal and electrically evoked noradrenaline overflow in the portal vein as well as on mean arterial pressure (MAP) and heart rate (HR) were investigated in permanently instrumented freely moving rats. R-α-Methylhistamine (0.01, 0.1 and 1 μmol/kg) inhibited the evoked noradrenaline overflow up to 43%, the ED₅₀ value being 0.013 μmol/kg. Thioperamide (0.1, 0.5 and 1.0 μmol/kg) antagonized the effect of 1.0 μmol/kg R-α-methylhistamine dose-dependently, evoked overflow returning to control values at 1.0 μmol/kg of the antagonist; thioperamide alone had no effect on electrically evoked noradrenaline overflow. Basal noradrenaline levels, blood pressure and heart rate were not at all influenced by R-α-methylhistamine and thioperamide, alone or in combination. The results clearly show the presence of prejunctional histamine H₃-receptors inhibiting the electrically evoked noradrenaline overflow from vascular sympathetic nerve terminals in the portal vein of freely moving rats. Received: 27 August 1996 / Accepted: 14 October 1996     

Characterization of presynaptic vascular muscarinic receptors inhibiting endogenous noradrenaline overflow in the portal vein of the freely moving rat.

1. In the portal vein of permanently cannulated, freely moving, unanaesthetized rats, methacholine (MCh) is able to inhibit the electrically-evoked endogenous noradrenaline (NA) overflow. This inhibition is mediated by presynaptic inhibitory muscarinic heteroreceptors. 2. By use of pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and AF-DX 116 as M1-, M3-, and M2-selective antagonists respectively, the MCh (0.1 microM)-induced inhibition of the electrically-evoked NA overflow could be reversed to the control stimulation value dose-dependently. 3. The potency order of the antagonists was: 4-DAMP greater than AF-DX 116 greater than pirenzepine, pIC50 values being 8.50, 7.96 and 7.01, respectively. 4. From these results it was concluded that the inhibitory presynaptic heteroreceptors in the portal vein of conscious unrestrained rats are of the cardiac M2-subtype.     

Intrinsic heart rate on exercise and the measurement of beta-adrenoceptor blockade

1 Methods of expressing the effects of β-adrenoceptor blocking drugs on exercise heart rate have been evaluated using a standardised exercise test.

2 In six normal subjects given atropine (0.04 mg/kg) on two separate occasions, the mean ± s.e. mean exercise heart rate rose by 10.3± 1.8 beats/min and by 11.0± 1.6 beats/min respectively. This increase was designated the `vagal effect and was not significantly different in the two studies.

3 After atropinsation, propranolol (0.2 mg/kg) reduced mean ± s.e. mean exercise heart rate by 45.3 ± 2.6 beats/min and 0.4 mg/kg by 50.8 ±4.5 beats/min. This mean sympathetic blockade was not altered significantly by increasing the dose of propranolol but, in four of the six subjects, the larger dose produced an increased effect of 4, 6, 12 and 16 beats/min, suggesting that maximum sympathetic blockade may not have been produced by 0.2 mg/kg.

4 Knowledge of the vagal effect in each subject with standardised exercise enabled prediction to be made of the exercise heart rate after propranolol (0.4 mg/kg) without previous atropinisation. Propranolol (0.4 mg/kg) was then given intravenously to each subject and the actual exercise heart rate measured. There was no significant difference between the predicted and observed exercise heart rates.

5 Propranolol (0.6 mg/kg) without atropine was then given to the four subjects who had shown increased effect with (0.4 mg/kg) and the sympathetic blockade was measured. In one subject, a further increase in sympathetic blockade of 10 beats/min was found.

6 The intrinsic heart rate at rest and on exercise was measured for propranolol (0.2 and 0.4 mg/kg) and, for propranolol (0.6 mg/kg), the intrinsic heart rate on exercise was calculated. At rest, although no significant difference was found between the two dose levels, three subjects did not appear to have maximum autonomic blockade at 0.2 mg/kg. Similarly, several subjects had lower intrinsic heart rates on exercise after 0.4 or 0.6 mg/kg than after 0.2 mg/kg. The intrinsic heart rate on exercise was significantly greater than that obtained at rest.

7 Using the maximum sympathetic blockade obtained in each subject as the sympathetic component of exercise, the effects of increasing oral doses of practolol on exercise heart were measured as percentage blockade of sympathetic effect and this was compared with other conventional methods of measuring β-adrenoceptor blockade. It was found that percentage blockade of sympathetic effect correlated most closely with both percentage and absolute reduction of exercise heart rate. Correlations with exercise heart rate after drug and percentage inhibition of tachycardia, whilst also significant, did not appear as good.

8 When the effects of practolol were expressed in terms of the potential blockade, a plateau occurred between 70 and 80% of `maximum' sympathetic blockade. The failure to achieve higher levels with practolol may be the result of its partial agonist or intrinsic sympathomimetic activity.

     

The uptake of noradrenaline by the isolated perfused rat heart

The uptake of noradrenaline by the isolated perfused rat heart was studied after perfusion with a medium containing various concentrations of (±)-[³H]-noradrenaline. Simultaneous measurement of the uptake of [³H]-noradrenaline and of the net increase in the noradrenaline content of the heart showed that [³H]-noradrenaline entering the heart both increased the tissue content and exchanged with endogenous noradrenaline. A large part (about 75%) of the endogenous noradrenaline pool, however, exchanged very slowly if at all with exogenous noradrenaline. The initial rates of noradrenaline uptake satisfied Michaelis-Menten kinetics with a Km for (±)-noradrenaline of 6.64×10^-7 M. Further analysis of the uptake process indicated that noradrenaline entered into at least two intracellular pools at different rates. Measurement of the initial rates of noradrenaline uptake during perfusion with various concentrations of nonradioactive (+)- and (-)-noradrenaline showed that the uptake process exhibited stereochemical specificity. The Km values for (+)- and (-)-noradrenaline were 13.9×10^-7 and 2.66×10^-7 M respectively. Cocaine acted as a potent competitive inhibitor of noradrenaline uptake. This finding suggested that diffusion did not play any significant role in the entry of noradrenaline into the tissue.     

Comparative alpha- and beta-adrenoceptor activity of 2- and 6-ring-chlorinated noradrenaline analogues

The alpha- and beta-adrenoceptor activity of the 2- and 6-ring-chlorinated analogues of noradrenaline (norepinephrine) were evaluated in vitro. The 2-chloro-substituted analogue exhibits a far greater affinity for beta 1-chronotropic receptors than the 6-chloro-substituted analogue, whereas no significant differences are apparent for their alpha-adrenergic affinities.     

Effects of prolonged ethanol administration on the noradrenaline levels of rat heart.

The effect of prolonged administration of ethanol on the noradrenaline concentration of the heart was investigated in rats. After 4 weeks there was no difference in the catecholamine levels between control and ethanol consuming rats. After 12 and 24 weeks there was a highly significant increase in heart noradrenaline concentration in hearts of experimental rats. These findings may indicate a delay in the effect of ethanol in cardiac tissue. It is further suggested that continued exposure to high levels of catecholamine may play a role in the development of cardiomyopathy in chronic alcoholism.     

Oxyhaemoglobin equilibrium and chronic beta-adrenoceptor blockade in coronary heart disease.

In 20 patients with coronary heart disease the effect of long-term beta-adrenergic receptor blockade on the haemoglobin oxygen equilibrium was investigated. Study patients received alprenolol 200 mg twice daily for 12-41 months (mean: 24 months) as a secondary preventive measure following a myocardial infarction. While on and again following gradual withdrawal of alprenolol, the patients performed a maximum bicycle ergometer test. Haemoglobin oxygen affinity as expressed by the P50 value, 2,3-diphosphoglycerate (2,3-DPG) and carbon monoxide haemoglobin were measured before and following exercise. Pre-exercise P50 decreased from 25.2 +/- 0.3 mm Hg (mean +/- s.e. mean) while on beta-adrenoceptor blocker to 24.6 +/- 0.4 mm Hg in the off-treatment state (P less than 0.05). Five minutes after stopping exercise P50 was 25.1 +/- 0.3 in patients taking alprenolol as compared to 24.7 +/- 0.3 after withdrawal of the drug (P less than 0.01). It is concluded that the slight decrease in haemoglobin oxygen affinity in long-term treatment with alprenolol, which is observed in the present study probably is without clinical bearing. The question should be further elucidated by analysis of coronary sinus blood samples.     

Effects of chlorpromazine on the isolated perfused rat heart.

Perfusion of hearts with chlorpromazine · HCl (50–5000 ng/ml) caused significant changes in heart rate, coronary flow, isometric systolic tension, certain metabolite concentrations, and electrical activity. Phosphorylase a activity remained normal. Chlorpromazine (50 ng/ml) decreased isometric systolic tension and prolonged QT interval in the electrocardiogram. Fructose 1,6-diphosphate and pyruvate concentrations were elevated, suggesting aldolase inhibition and decreased pyruvate utilization. Similar results were obtained at 500 ng/ml. At 5000 ng/ml, heart rate and isometric systolic tension were markedly diminished. Coronary flow increased briefly but returned to normal. PR, QT_a, and QT intervals were prolonged. Fructose 1,6-diphosphate, glyceraldehyde-3-phosphate, and pyruvate concentrations in the tissue increased, while the amount of dihydroxyacetone phosphate and l-(?)-glycerol-1-phosphate decreased. No chlorpromazine-induced lesions, gross or microscopic, were found.