Interactions of angiotensin and cocaine on the output of noradrenaline from isolated rabbit hearts

Summary The output of noradrenaline from isolated rabbit hearts during sympathetic nerve stimulation is increased by angiotensin (1.3 ng/ml) to 176% of the preceding control stimulation period. During inhibition of noradrenaline re-uptake by cocaine (5 or 15 g/ml), the augmentation caused by the peptide is unchanged (181 and 171%, respectively). The result favours the assumption that angiotensin enhances the output of noradrenaline by an increase of the amount of transmitter liberated from the nerve terminals rather than by interfering with transmitter inactivation.     

Influence of drugs with affinity for α-adrenoceptors on noradrenaline release by potassium,tyramine and dimethylphenylpiperazinium

The influence of oxymetazoline and phentolamine on the overflow of noradrenaline evoked by potassium, tyramine and dimethylphenylpiperazinium (DMPP) was investigated in isolated perfused rabbit hearts.Oxymetazoline decreased, and phentolamine increased, the outflow of noradrenaline evoked by potassium. In hearts previously perfused with (?)-³H-noradrenaline, oxymetazoline reduced, and phentolamine enhanced, potassium-induced overflow of both ³H-noradrenaline and total tritium. These actions closely resemble previously described effects on noradrenaline overflow evoked by electrical stimulation of sympathetic nerves. At concentrations which modified the response to potassium, oxymetazoline and phentolamine did not influence the overflow evoked by tyramine. Both drugs diminished DMPP-induced overflow.It is concluded that oxymetazoline depresse noradrenaline release evoked by potassium or orthodromic action potentials through activation of neuronal α-adrenoceptors, followed by inhibition of electro-secretory coupling. Phentolamine blocks the analogous inhibitory effect of liberated noradrenaline and thus enhances release. The action of tyramine does not involve electro-secretory coupling and therefore is not changed. The influence of oxymetazoline and phentolamine on noradrenaline release by DMPP is not related to α-adrenoceptors.     

The effect of methacholine on noradrenaline release from the rabbit heart perfused with indometacin

Summary The experiments were undertaken in order to study the effect of inhibition of prostaglandin synthesis on the muscarinic inhibition of noradrenaline release evoked by sympathetic nerve stimulation. The right sympathetic nerves of the perfused rabbit heart were stimulated electrically. The noradrenaline output was enhanced after perfusion of the hearts with indometacin 3×10^–5 M indicating blockade of the prostaglandin-mediated negative feedback control. Both in the presence and in the absence of indometacin methacholine 4×10^–5 M decreased the noradrenaline output by a similar percentage. It is concluded that the muscarinic inhibition of noradrenaline release does not require the functional integrity of the prostaglandin-mediated feedback system.     

Differential effects of hypothermia on neuronal efflux,release and uptake of noradrenaline

Summary 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.     

The effects of acute administration of propranolol and practolol on the uptake,content, release,and turnover of noradrenaline in the rat heart in vivo

Summary The effect of the acute administration of (±)-, (+)-, and (–)-propranolol and practolol, respectively, on the cardiac turnover of noradrenaline was studied in male Wistar rats kept under controlled conditions of environmental lighting. Propranolol caused no or only minor effects on the turnover in concentrations of 0.001–0.1 mmoles/kg, whereas higher doses (0.2–0.4 mmoles/kg) decreased the noradrenaline turnover in rat hearts concomitantly with toxic signs from the CNS. The endogenous noradrenaline content was not changed by any concentration of propranolol. Practolol (0.1–0.4 mmoles/kg) did not influence the cardiac noradrenaline turnover, but, like an indirectly acting sympathomimetic drug, released noradrenaline.Supported by a grant of the Deutsche Forschungsgemeinschaft.     

ACTH increases noradrenaline release in the rabbit heart

Summary The effects of ACTH on the release of noradrenaline and the increase of heart rate produced by sympathetic nerve stimulation (1 Hz) were studied in isolated perfused rabbit hearts. ACTH-(1–24) 0.1–100 nmol/l increased the stimulation-evoked overflow of noradrenaline concentration-dependently, reversibly and up to two-fold. The basal outflow of noradrenaline, the basal heart rate and the stimulation-evoked increase in heart rate were not changed. Human ACTH-(1–39) also increased the evoked overflow of noradrenaline. The effect of ACTH-(1–24) 0.3 nmol/l persisted after blockade of -adrenoceptors with propranolol and blockade of neuronal catecholamine uptake by cocaine. ACTH-(1–24) 3 nmol/l did not change the removal of noradrenaline from the perfusion fluid, when hearts were perfused with medium containing 59 nmol/l noradrenaline. The results show that ACTH increases the action potential-evoked release of noradrenaline from cardiac postganglionic sympathetic neurones, probably by activating specific presynaptic ACTH receptors. The high potency of ACTH suggests that these presynaptic receptors may be activated in vivo by circulating ACTH under certain pathophysiological conditions.Send offprint requests to B. Szabo at the above address     

Time-dependent changes in neuronal net uptake of noradrenaline after pretreatment with pargyline and/or reserpine

Summary Isolated rabbit hearts were perfused with 20 to 200 ng/ml of (–)-noradrenaline and arterio-venous differences were determined at various times to measure the rate of net removal of the amine from the perfusion fluid. Animals were untreated or pretreated with reserpine and/or pargyline to block vesicular retention and/or intraneuronal monoamine oxidase (MAO).The arterio-venous difference (in percent of the arterial concentration) remained rather constant during prolonged perfusions of untreated hearts with (–)-noradrenaline, the magnitude of the difference being inversely related to the arterial concentration. After block of MAO the rate of net removal declined exponentially with time; the rate of decline increased with increasing arterial concentration of the amine and also after the additional pretreatment with reserpine. The time-dependent decline in the rate of net removal was shown to be due to an increased efflux of the amine from the nerve endings. The net removal of noradrenaline-H³ at the 5th min of perfusion of pargyline-pretreated hearts was mainly due to neuronal net uptake, since a) O-methylation accounted for only 5% of the removal, and b) cocaine (10–30 (g/ml) virtually abolished net removal.Initial rates of removal were not affected by the various pretreatments.In untreated hearts retention of exogenous (–)-noradrenaline increased linearly with the duration of the perfusion but the increase was exponential after block of MAO. Apparently, the storage capacity becomes exhausted during prolonged perfusions of pargyline-pretreated hearts.The ratio noradrenaline retained by the heart/noradrenaline removed by the heart was quite small in untreated (0.16), very small in reserpine-pretreated (0.03) and nearly unity in pargyline-pretreated hearts.It is concluded that any impairment of the intraneuronal mechanisms of inactivation (vesicular storage and MAO) leads to an increase in the axoplasmic concentration of free noradrenaline which causes an increased efflux of the amine, while the influx remains unchanged. The axoplasmic concentration of free noradrenaline seems to rise more after block of MAO than after pretreatment with reserpine and is most pronounced after both. Changes in the sensitivity of the pacemaker to (–)-noradrenaline were found to be correlated with changes in the rate of removal of the amine from the perfusion fluid.Part of this study was supported by the Deutsche Forschungsgemeinschaft.     

Central action of phentolamine administered intraventricularly in the rat

Phentolamine injected intraventricularly in unanaesthetized rats decreased their locomotor activity and antagonized the excitatory effect of intraventricularly injected noradrenaline, or amphetamine injected s.c. Phentolamine did not influence the level of noradrenaline and 5-hydroxytryptamine in the rat brain. The mechanism of observed action of phentolamine is discussed.     

Effects of hypoxia on noradrenaline release and neuronal reuptake in isolated rabbit thoracic aortic strips

Summary To clarify the effects of hypoxia on stimulus-evoked noradrenaline release and on neuronal reuptake of the released noradrenaline, we examined the effects of hypoxia on contraction responses of rabbit thoracic aortic strips to transmural electrical stimulation and on the stimulation-evoked overflow of total [³H] and [³H]noradrenaline from the strips prelabelled with [³H]noradrenaline. This was done in the presence or absence of an inhibitor of neuronal uptake (cocaine). In a medium equilibrated with a gas mixture of 95% O₂/5% CO₂ (control), cocaine doubled the stimulation-evoked overflow of total [³H] and [³H]noradrenaline; there was a concomitant increase (130%) in contractions to electrical stimulation. At 0% O₂ (95% N₂/5% CO₂, hypoxia), cocaine had no significant effects on either the stimulation-evoked overflow of total [³H] and [³H]noradrenaline or contractions. In the absence of the drug, hypoxia decreased the stimulation-evoked overflow of total [³H] and [³H]noradrenaline to 47% and 43%, respectively, of the control values, whereas these values were 31% and 28%, respectively, after exposure to cocaine. The inhibition by hypoxia of contraction responses to electrical stimulation was greater in the presence of cocaine than in its absence. These results show that hypoxia inhibits both noradrenaline release evoked by a given stimulus and neuronal uptake.This work was supported by a Grant-in-Aid for New Drug Development from the Ministry of Health and Welfare of Japan and by a Grant from Smoking Research Foundation, Japan Send offprint requests to S. Miwa at the above address     

Effects of 4-aminopyridine on mechanical activity and noradrenaline release in the rat portal vein in vitro.

4-Aminopyridine (4-AP) increased the spontaneous mechanical activity of the isolated rat portal vein. Since denervation and adrenergic receptor blockade failed to prevent this effect of 4-AP it is suggested that the drug enhances the electrical excitability of the muscle membrane. 4-AP significantly increased the response of the muscle to electrical nerve stimulation in most experiments but had little effect on the response to applied noradrenaline (NA). Both spontaneous and evoked release of 3H-activity, following preincubation in 3H-noradrenaline, were increased in the presence of 4-AP (10(-3) M). The present results with 4-AP can be explained by its known ability to block the transient potassium conductance which accompanies the action potential in excitable tissues.     

Evidence for the presynaptic location of the alpha-adrenoceptors which regulate noradrenaline release in the rat submaxillary gland

Summary Fifteen days after duct ligation, the wet weight of the rat submaxillary gland was reduced to 40% of the contralateral control. Under these experimental conditions, the noradrenaline (NA) content expressed as g/g was 1.2±0.1 in the control glands and 1.9±0.2 in the atrophied glands.The accumulation of ³H-NA in the tissue expressed as Ci/gland, did not differ when the atrophied glands were compared with the corresponding controls. Consequently, the uptake and retention of ³H-NA was not modified by the atrophy of the secretory cells of the gland.The spontaneous efflux of radioactivity from normal and atrophied submaxillary glands prelabelled with ³H-NA was similar. The analysis of the metabolic pattern in both experimental groups revealed that in the spontaneous outflow and also during potassium-induced depolarization, the formation of the O-methylated metabolite, ³H-normetanephrine (NMN) was reduced by more than 50% in the atrophied glands. During depolarization induced by K⁺, a 2-fold increase in the outflow of the deaminated glycol ³H-3,4-dihydroxyphenylglycol (DOPEG) was observed.The effect of phentolamine on the release of radioactivity induced by 60 mM K⁺ in normal and in atrophied submaxillary gland slices prelabelled with ³H-NA was also investigated. In both experimental groups, the fractional release of total radioactivity induced by K⁺ was similar. Phentolamine, 3.1 M, produced a 3-fold increase in the fractional release of radioactivity both in the control and the atrophied glands. These results indicate that the increase of K⁺-induced release of ³H-NA induced by phentolamine was independent of the presence or absence of the postsynaptic structures. It is concluded that phentolamine increases transmitter release by blocking alpha-adrenoceptors located in the noradrenergic nerve endings of the rat submaxillary gland.     

Regulation of adrenergic receptor number following chronic noradrenaline infusion in the rabbit

In order to study noradrenaline-induced regulation of alpha- and beta-adrenoceptors, groups of male New Zealand White rabbits (n = 8) were treated with intravenous noradrenaline (0.09 mol/kg × h) or ascorbate (0.1 %) for I0 days via osmotic minipumps implanted in the femoral vein, and the number of cardiac, lung and lymphocyte beta-adrenoceptors as well as renal and platelet alpha₂-adrenoceptors were determined.

Binding of exogenous noradrenaline by the proteins of dog plasma

Summary On addition to dog blood or plasma about 40% of exogenous nor-adrenaline escaped detection by photofluorimetric methods, when noradrenaline was present in concentrations ranging from 10 to 1000 ng/ml. As shown by bioassay the missing noradrenaline was not inactivated, but rather bound to plasma proteins; this was shown by experiments using labelled noradrenaline and precipitation of proteins, or Sephadex gel filtration. Cellulose acetate electrophoresis demonstrated binding by all protein fractions, alpha 1 and 2 globulins showing the greatest avidity for noradrenaline. Drugs known to be highly bound by proteins did not affect the binding capacity for noradrenaline. It is concluded that plasma noradrenaline values found after injection or infusion of noradrenaline may be only about half of the real values.Supported by Instituto de Alta Cultura (Research Project PMC-2).     

Mathematical modelling and quantification of the autoinhibitory feedback control of noradrenaline release in brain slices

Summary Concentration-response curves, reflecting ₂-autoreceptor-mediated inhibition of [³H]-noradrenaline release by exogenous noradrenaline in rat cerebral cortex and rabbit hippocampus slices, were analysed in order to test the usefulness of a mathematical model describing the relation between the independent variable, exogenous noradrenaline, and the dependent variable, inhibition of release. This model was based on the assumption of direct proportionality between receptor occupation and response, implying that there is correspondence between the shape of a concentration-binding curve and a concentration-response curve. The experimental concentration-response curves were obtained by different approaches: noradrenaline release from brain slices prelabelled with [³H]-noradrenaline was elicited electrically either by pseudo-one-pulse (POP) stimulation or by stimulation with 36 pulses applied with a frequency of 3 Hz. POP stimulation avoids autoinhibition by released noradrenaline and, therefore, was a suitable touchstone for the applied mathematical model which evaluates by nonlinear regression analysis two primary parameters: the dissociation constant between noradrenaline and the ₂-adrenoceptor and the biophase concentration of noradrenaline which reflects the extent of autoinhibition and should be zero under POP conditions. In rat cerebral cortex tissue, the corresponding biophase concentration of endogenous noradrenaline was indeed estimated to be zero and the dissociation constant was K _d = 10^{–7.62±0.14} mol/l. With 3 Hz stimulation, the biophase concentration was 10^{–7.80±0.05} mol/l, which has to be interpreted with respect to a simultaneously estimated K _d of 10^{–7.63±0.12} mol/l. Since the K _d-values under POP or 3 Hz conditions were similar, the biophase concentration obviously had no influence on the estimate of the other primary parameter, K _d. With rabbit hippocampus, however, the main prerequisite of the mathematical model; i.e. direct proportionality between receptor occupation and response, was not established since the slope of the POP concentration-response curve (estimated as slope parameter c) did not correspond to that of a concentration-binding curve.In conclusion, mathematical modelling by nonlinear regression analysis of the autoinhibitory circuit of noradrenaline release allows the estimation of a parameter c of this feedback regulation which supports or rejects the assumption of direct proportionality between receptor occupation and functional response. When the given requirement of direct proportionality is shown to hold, this analysis allows the feedback circuit to be described quantitatively in terms of the affinity of noradrenaline for, and of the biophase concentration of noradrenaline at, the presynaptic ₂-adrenoceptors.Correspondence to T. J. Feuerstein     

酚妥拉明联合垂体后叶素治疗支气管扩张咯血的疗效观察

目的探讨酚妥拉明联合垂体后叶素治疗支气管扩张咯血的临床疗效。方法将72例患者随机分为观察组和对照组（各36例）,对照组在综合性基础治疗上给予垂体后叶素治疗,观察组在对照组治疗的基础上加用酚妥拉明治疗。结果观察组总有效率为97．2％,明显优于对照组的77．8％,两组总有效率比较差异具有统计学意义（P〈0．05）。结论应用酚妥拉明联合垂体后叶素治疗支气管扩张咯血疗效显著,具有起效快、安全可靠、不良反应少等优点,值得临床推广。     

Metabolism of endogenous and exogenous noradrenaline in the rabbit perfused heart

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from rabbit perfused hearts was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs and without nerve stimulation, the outflow of endogenous noradrenaline over a period of 108 min averaged 0.17 pmol×g^–1×min^–1 and the outflow of DOPEG 2.1 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.13 pmol×g^–1×min^–1). The effect of perfusion with (–)-noradrenaline 0.1, 1 or 10 mol/l for 18 min was then investigated. As the concentration of noradrenaline increased so did the outflow of DOPEG. Moreover, DOMA was found in the venous effluent during and after perfusion with noradrenaline 1 or 10 mol/l. The increase in the outflow of DOPEG and DOMA was almost abolished when cocaine 10 mol/l was present during the perfusion with noradrenaline 1 mol/l. The release of endogenous noradrenaline by sympathetic nerve stimulation or tyramine 10 mol/l, but not the release evoked by nicotine 30 mol/l, was accompanied by an increase in the outflow of DOPEG; an outflow of DOMA was not observed.It is concluded that, in the rabbit perfused heart, DOPEG is an important metabolite of endogenous noradrenaline. DOMA is at best a minor product, either when the neurones are at rest or when noradrenaline is released by sympathetic nerve stimulation, nicotine or tyramine. DOMA is formed in detectable amounts when the tissue is exposed to a high concentration of exogenous noradrenaline. Like DOPEG, it is formed intraneuronally. The results confirm and extend those obtained previously on guinea-pig incubated atria. They make it unlikely that, in these tissues at least, DOMA formation is one of the physiological pathways of noradrenaline catabolism.     

Polymyxin B,a selective inhibitor of protein kinase C,diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

Summary Slices of the rabbit hippocampus were labelled with ³H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.Abbreviations PKC protein kinase C - PDB phorbol 12,13-dibutyrate - TPA 12-O-tetradecanoyl 13-acetate     

Electrically induced release of endogenous noradrenaline and dopamine from brain slices: pseudo-one-pulse stimulation utilized to study presynaptic autoinhibition

Summary Slices of rat hypothalamus (noradrenaline experiments) or rabbit caudate nucleus (dopamine experiments) were prepared, superfused, and field-stimulated using series of monophasic rectangular pulses. Noradrenaline, dopamine and the main dopamine metabolite, dihydroxyphenylacetic acetic acid (DOPAC), were determined using HPLC with electrochemical detection. Electrical stimulation was performed using the following protocols: 1) 4 pulses delivered at 100 Hz; this type of stimulation is referred to as pseudo-one-pulse stimulation (POP); its short duration of only 32 ms does not allow the development of autoinhibition; 2) 2 bursts of 4 pulses at 100 Hz, delivered 1 s apart (2-POP-stimulation); 3) 8 pulses at 1 Hz (dopamine experiments only); 4) 36 pulses at 3 Hz. Noradrenaline experiments. The ₂-adrenoceptor antagonist yohimbine (1 mol/l) did not enhance noradrenaline overflow following POP stimulation, but enhanced the overflow following 2-POP-stimulation by about 50% and that following 36-pulse-stimulation by almost 100%. Dopamine experiments. The D₂-dopamine receptor antagonist sulpiride (3 mol/l) facilitated the overflow of dopamine elicited with 2-POP-stimulation (66%), 8 pulses/1 Hz (92%), and 36 pulses/3 Hz (140%). It did not significantly facilitate the overflow of dopamine following POP-stimulation (19%). The overflow of DOPAC was not, or only slightly, increased by electrical stimulation, and its spontaneous outflow was more than three times higher than that of dopamine. Furthermore, the electrically induced overflow of dopamine did not exceed the outflow of DOPAC at any of the stimulation conditions employed.The results of the present study bear out important claims of the autoreceptor theory and confirm the data obtained in previous experiments using labelled transmitters. Correspondence to E. A. Singer at the above address     

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.