Effects of the Aconitum alkaloid mesaconitine in rat hippocampal slices and the involvement of α- and β-adrenoceptors

1. The effects of mesaconitine, the main alkaloid contained in Aconiti tuber, were investigated by use of extracellular recordings of stimulus-evoked population spikes and field excitatory postsynaptic potentials (e.p.s.ps) in the CA1 region of rat hippocampal slices.
2. At a concentration of 10 nM, mesaconitine evoked excitations, which were manifested as an increase in the amplitude of the orthodromic spike and the appearance of multiple spikes following the first postsynaptic spike, without affecting the magnitude of paired-pulse facilitation. The increase in spike amplitude was persistent and was not reversed by up to 90 min of washout. At concentrations of 30 and 100 nM, the alkaloid produced a biphasic effect, that is an excitation followed by an inhibition without having any effect upon the field e.p.s.p. At concentrations above 100 nM, mesaconitine suppressed the orthodromic population spike and the field e.p.s.p.
3. The excitatory effect was also observed when electrical stimulation was stopped completely during the application of mesaconitine (10 nM) and during the first 15 min of washout.
4. The enhancement of the population spike and the appearance of multiple spikes induced by mesaconitine (10–100 nM) were blocked by pretreatment with the β-adrenoceptor antagonists propranolol (1 μM) and timolol (1 μM), whereas the inhibitory effect was blocked by the α-adrenoceptor antagonists yohimbine (1 μM) and phentolamine (10 μM). However, when the β-adrenoceptor antagonist timolol was added 10 min after the application of mesaconitine, it failed to block the long-lasting enhancement of the spike amplitude and the appearance of multiple population spikes.
5. Application of the selective β-adrenoceptor agonist isoprenaline (500 nM) to the hippocampal slices induced an increase in the amplitude of the orthodromic population spike and elicited 2–3 additional spikes. Mesaconitine (10 nM) did not further potentiate this enhancement of the spike amplitude when added after a 15 min pretreatment with isoprenaline.
6. Perfusion of forskolin, which directly activates adenylate cyclase, enhanced the population spike. Mesaconitine had no additional effect when applied after pretreatment with forskolin.
7. It is concluded that the excitatory effects evoked by lower concentrations of the plant alkaloid mesaconitine are mediated by stimulation of β-adrenoceptors and the consequent activation of intracellular processes which lead to the long-lasting changes in excitability.

     

The effectiveness of β-adrenoceptor stimulation and the contribution of β1-adrenoceptors increase from the proximal to the distal part of the canine saphenous vein

Summary The present study was undertaken to characterize -adrenoceptors of the canine saphenous vein and their distribution along this vessel. In a first series of experiments, concentration-response curves to isoprenaline and forskolin were compared on strips taken from proximal and distal portions of the vein. The tone of the strip was previously increased by phenylephrine to one of the three levels: about 85, 70 and 55% of the maximum, which corresponds to 3.08±0.16 and 2.96±0.17 (n = 6) N·g^–1, for proximal and distal strips, respectively. The maximal relaxation to isoprenaline was significantly larger in the distal than in the proximal portion, for responses starting at 85 and 70% of the maximum tone. In contrast, forskolin caused 100% relaxation, both proximally and distally, irrespective of the previous tone.In a second series of experiments, the relaxation to dobutamine and terbutaline was compared in proximal and distal portions after the tone had been elevated by adrenaline, noradrenaline and phenylephrine to about 70% of the maximum. When the tone was increased by adrenaline, the relaxation in response to 2.7 mol·l^–1 dobutamine was larger than that to 2.7 mol·l^–1 terbutaline both in proximal and distal portions, while when it was increased by noradrenaline, the relaxation to terbutaline was larger than that to dobutamine in the proximal portion; in the distal portion, dobutamine and terbutaline were equieffective. When the tone was increased by phenylephrine, dobutamine and terbutaline caused equivalent relaxations proximally, but distally the relaxation to dobutamine was larger than that to terbutaline.In a third series of experiments, the antagonism exerted by atenolol and by the compound ICI-118,551 on the relaxation to dobutamine and terbutaline was compared proximally and distally. Both proximally and distally atenolol was more effective against dobutamine than against terbutaline, while the compound ICI-118,551 was effective only against terbutaline.It is concluded that ₁- and ₂-adrenoceptors exist in the canine saphenous vein and that the effectiveness of -adrenoceptor stimulation increases from the proximal to the distal portion of this vessel due to a progressive increase in the density of ₁-adrenoceptors.Correspondence to S. Guimaraes at the above address     

The involvement of α2-adrenoceptors in the anticonvulsive effect of swim stress in mice

RATIONALE: Various studies have shown that stressful manipulations in rats and mice lower the convulsant potency of GABA-related, but also some GABA-unrelated convulsants. The mechanism of this anticonvulsive effect of stress is still unknown. OBJECTIVES: We tested the possible involvement of alpha2-adrenoceptors in the previously observed anticonvulsive effect of swim stress. METHODS: The mice were, prior to exposure to swim stress and the IV infusion of picrotoxin, pre-treated with clonidine (an alpha2-adrenoceptor agonist), yohimbine (a non-selective alpha2-adrenoceptor antagonist), idazoxan (a selective alpha2-adrenoceptor antagonist), or niguldipine (an alpha1-adrenoceptor antagonist), and the latency to the onset of two convulsant signs was registered. RESULTS: In control unstressed animals clonidine (0.1 and 1 mg/kg IP), yohimbine (2 mg/kg IP) and idazoxan (1 mg/kg IP) failed to affect the doses of picrotoxin needed to produce convulsant signs, while niguldipine (5 mg/kg IP) prolonged the latency, i.e. it enhanced the doses of picrotoxin producing running/bouncing clonus and tonic hindlimb extension. In swim stressed mice clonidine enhanced, while idazoxan decreased doses of picrotoxin needed to produce two convulsive signs. Yohimbine decreased the dose of convulsant needed to produce tonic hindlimb extension, while niguldipine enhanced doses of picrotoxin needed to produce both symptoms. CONCLUSIONS: The results demonstrate the alpha2-adrenoceptor agonist-induced potentiation and alpha2-adrenoceptor antagonist-induced diminution of the anticonvulsive effect of stress. Additionally, they show the anticonvulsive effect of niguldipine in unstressed and stressed animals. Hence, the results suggest that alpha2-adrenoceptors are involved in the anticonvulsive effect of swim stress in mice.     

Presynaptic β2-adrenoceptors on the sympathetic nerve fibres of the human saphenous vein: no evidence for involvement in adrenaline-mediated positive feedback loop regulating noradrenergic transmission

Summary Spirally cut strips of human saphenous veins preincubated with ³H-noradrenaline were superfused in the presence of corticosterone and, unless stated otherwise, of cocaine or desipramine. Tritium overflow was stimulated electrically (2 Hz). Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential ₂-adrenoceptor agonist procaterol concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a -adrenoceptor agonist with moderate preference for ₁-adrenoceptors, was ineffective. The concentration-response curve of isoprenaline was shifted to the right by the nonselective -adrenoceptor antagonist propranolol and by the preferential ₂-adrenoceptor antagonist ICI 118,551, but was not affected by the ₁-selective antagonist atenolol. In experiments on strips preexposed to adrenaline 10 nmol/l (i. e. a concentration higher than that which normally occurs in vivo) for 32 min in the absence of cocaine or desipramine, the electrically evoked ³H overflow was not affected 12 and 44 min after withdrawal of adrenaline, irrespective of whether propranolol was absent or present in the superfusion fluid. — In veins incubated with ³H-adrenaline, a considerable amount of the radioactivity was accumulated. During subsequent superfusion with ³H-adrenaline-free solution, electrical stimulation induced tritium overflow in a tetrodotoxin-sensitive manner. Propranolol failed to modify the evoked tritium overflow. — It is concluded that the sympathetic nerve fibres of the human saphenous vein are endowed with facilitatory presynaptic ₂-adrenoceptors. These receptors do not seem to play a substantial role in a local adrenaline (previously taken up)-mediated positive feedback loop regulating noradrenergic transmission, at least under the present in vitro conditions.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert     

Distinct down-regulation of cardiac β1- and β2-adrenoceptors in different human heart diseases

Summary Cardiac -adrenoceptor density and ₁- and ₂-subtype distribution were examined in human left ventricular myocardium from transplant donors serving as controls and from patients with mitral valve stenosis, aortic valve stenosis, idiopathic dilated cardiomyopathy, and ischaemic cardiomyopathy respectively. The total -adrenoceptor density was similar in transplant donors and patients with moderate heart failure (NYHA II–III) due to mitral valve stenosis, but was markedly reduced in all forms of severe heart failure (NYHA III–IV) studied. A reduction of both ₁- and ₂-adrenoceptors was found in patients with severe heart failure due to mitral valve stenosis or ischaemic cardiomyopathy. In contrast, a selective down-regulation of ₁-adrenoceptors with unchanged ₂-adrenoceptors and hence a relative increase in the latter was observed in idiopathic dilated cardiomyopathy and aortic valve stenosis. It is concluded that the extent of total -adrenoceptor down-regulation is related to the degree of heart failure. Selective loss of ₁-adrenoceptors is not specific for idiopathic dilated cardiomyopathy but also occurs in aortic valve stenosis. Changes in ₁- and ₂-subtype distribution are rather related to the aetiology than to the clinical degree of heart failure. Send offprint requests to M. Steinfath at the above address     

Potentiation of the relaxing action of isoproterenol by forskolin in rabbit aortic rings: The involvement of β2-adrenoceptors

• 1.1. Forskolin, an activator of adenylate cyclase, potentiated the relaxing response to isoproterenol in rabbit aortic rings precontracted by phenylephrine (PE).
• 2.2. The potentiating effect of forskolin was inhibited by propranolol, a β-adrenoceptor inhibitor, but not by methylene blue, a guanylate cyclase inhibitor.
• 3.3. The relaxing response to terbutaline, a β₂-adrenoceptor agonist, but not lower concentrations of dobutamine, a β₁-adrenoceptor agonist, also was potentiated by forskolin. Forskolin, however, potentiated the relaxing response to high concentrations of dobutamine, which activates both β₁- and β₂-adrenoceptors.
• 4.4. Yohimbine, an α₂-adrenoceptor inhibitor, glyburide, an ATP-sensitive K⁺ channel inhibitor, iberiotoxin, a Ca²⁺-activated K⁺ channel inhibitor, or endothelium-removal failed to affect the potentiating effect of forskolin.
• 5.5. Dibutyryl cyclic AMP (cAmp) also potentiated the relaxing response to terbutaline.
• 6.6. These results suggest that in rabbit aortic rings forskolin causes the apparent potentiation of isoproterenol-induced relaxation by mainly affecting the relaxing response due to the activation of β₂-adrenoceptors by the forskolin-induced increase in the level of cAMP.

     

Effect of thyroid status on β-adrenoceptors and calcium channels in rat cardiac and vascular tissue

Summary To determine the influence of thyroid hormone on -adrenoceptors and Ca²⁺ channels, rats were treated with thyroxine (75 g/100 g sc daily for 5 days) or propylthiouracil (0.05% in drinking water for 30 days). -Adrenoceptor density in ventricular tissue, measured by [¹²⁵I]iodocyanopindolol binding, was significantly increased and decreased respectively, following thyroxine or propylthiouracil treatment to 124.7 ± 7.11 fmol/mg protein and 71.98 ± 5.37 fmol/mg protein from euthyroid (control) levels of 93.7 ± 4.58 fmol/mg protein. Ca²⁺ channel density, measured by [³H]nitrendipine binding, was altered in the opposite direction; it was significantly decreased and increased to 324 ± 24 fmol/mg protein and 691 ± 31 fmol/mg protein from 562 ± 35 fmol/mg protein after thyroxine or propylthiouracil treatment, respectively. No changes in affinity of either ligand were observed. Responses of isolated papillary muscles from propylthiouracil-treated animals accorded with changes seen in the binding studies. The geometric mean EC₅₀ of isoproterenol increased from 9.5 × 10^–9 mol/1 to 5.5 × 10^–8 mol/l, and the EC₅₀ for calcium decreasedfrom 3.16 × 10^–3 mol/1to 1.36 × 10^–3 mol/1; moreover, the responsiveness to the Ca ²⁺ channel activator Bay K 8644 was increased. The corresponding responses in thyroxine-treated animals could not be examined because of prominent arrhythmic activity. As with papillary muscles the sensitivity of left atria to isoproterenol was decreased after treatment with propylthiouracil, with geometric mean EC₅₀ values increasing from 3.21 × 10^–9 mol/1 to 89.4 × 10^–9 mol/l. This was however, associated with a decrease in the sensitivity to calcium with geometric mean EC₅₀ values increasing from 2.43 × 10^–3 mol/1 to 5.33 × 10^–3 mol/1 and a reduction in the maximal response to Bay K 8644. Treatment with thyroxine had no effect on tissue sensitivity. The responses of isolated tail artery to phenylephrine, calcium and Bay K 8644 were not significantly different in propylthiouracil- or thyroxine-treated animals. Send offprint requests to D. J. Triggle at the above address     

Effects of κ-opioid receptor stimulation in the heart and the involvement of protein kinase C

1. The role of protein kinase C (PKC) in mediating the action of κ-receptor stimulation on intracellular Ca²⁺ and cyclic AMP production was determined by studying the effects of trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte.
2. U50,488H at 2.5–40 μM decreased both the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the κ-agonist, that were blocked by a selective κ-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ-receptor stimulation.
3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca²⁺]_i transient induced by caffeine, that depletes the SR of Ca²⁺, was used as an indicator of Ca²⁺ content in the SR. The caffeine-induced [Ca²⁺]_i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine-induced [Ca²⁺]_i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca²⁺ from the SR. When the increase in IP₃ production in response to κ-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ-receptor stimulation does not involve IP₃. The observations suggest that PKC may act directly at the SR.
4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ-receptor stimulation on Ca²⁺ in the SR and cyclic AMP production, both of which play an essential role in Ca²⁺ homeostasis in the heart.

     

Differential effects of electrical stimulation,blockade of neuronal amine uptake and activation of α2-adrenoceptors on the release of endogenous noradrenaline and 5-hydroxytryptamine from the isolated rat pineal gland

Summary Isolated rat pineal glands were incubated in vitro and the release of endogenous noradrenaline or 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection. In the absence of test drugs, the spontaneous outflow of noradrenaline was about 10 fmol/10 min and electrical stimulation (5 Hz, 1500 pulses) evoked the release of about 70 fmol noradrenaline. Nomifensine enhanced the spontaneous outflow of noradrenaline about threefold and the electrically evoked release of noradrenaline about sixfold. In the presence of nomifensine, the ₂-adrenoceptor antagonist yohimbine markedly increased the electrically evoked release of noradrenaline, whereas the ₁-adrenoceptor antagonist prazosin had no effect. Clonidine inhibited the electrically evoked release of noradrenaline by about 65%, and this was antagonized by yohimbine in a competitive manner. In the absence of drugs, the initial spontaneous outflow of 5-HT was (compared with noradrenaline) very high 64 mol/10 min. It declined by 80% within 1 h of incubation in vitro. The outflow of 5-HIAA amounted initially to 38 mol/10 min and declined by 40% within 1 h of incubation. Addition of l-tryptophan (10 mol/1) after 1 h of incubation in vitro largely enhanced the outflow of 5-HT and 5-HIAA within 30 min of incubation (about ten- and fourfold, respectively). When l-tryptophan was present from the onset of incubation the initial outflow of 5-HT and 5-HIAA was only slightly elevated, but the decline was largely attenuated. Neither omission of calcium nor addition of nomifensine, clonidine or yohimbine significantly affected the spontaneous outflow of 5-HT or 5-HIAA. Likewise, neither electrical stimulation in the absence or presence of nomifensine and yohimbine nor stimulation by high potassium (45 mmol/1) significantly affected the outflow of 5-HT or 5-HIAA.In conclusion, the release of endogenous noradrenaline from the sympathetic nerves terminating in the pin eal gland is inhibited by presynaptic ₂-adrenoceptors. The outflow of 5-HT from the pineal gland originates almost exclusively from non-neuronal cells, most probably the pinealocytes, and depends largely on a continuous de novo synthesis. Catabolism of 5-HT to 5-HIAA in the pineal gland occurs mainly in an extraneuronal compartment, probably the pinealocytes and/or the interstitial cells of the pineal gland. Send offprint requests to K. Racké at the above address     

Different steric characteristics of β1- and β2-adrenoceptors

Summary -Adrenoceptors of lung (75% ₂) and heart (95% ₁) of calf were labelled with ³H-(–)-propranolol. The stereoisomers of 10 ligands were used to inhibit the binding of ³H-(–)-propranolol to membrane particles. The affinity ratio of sereoisomers is consistently greater for ₁-adrenoceptors than for ₂-adrenoceptors, regardless of whether the ligands are agonists, partial agonists or antagonists. The ₁-adrenoceptor appears to possess stricter steric requirements than the ₂-adrenoceptor. This property may prove helpful in differentiating the -adrenoceptor subtypes during receptor solubilization and purification.     

Regional distribution of β1- and β2-adrenoceptors in the failing and nonfailing human heart

Summary Total -adrenoceptor density and ₁- and ₂-subtype distribution in right and left atria and in different ventricular regions from 14 failing and seven nonfailing human hearts have been compared. End-stage heart failure was due to idiopathic dilated cardiomyopathy (n=8) or ischaemic cardiomyopathy (n=6).In nonfailing hearts the total -adrenoceptor density was similar in the right and left atria and in all the ventricular regions studied (about 70 to 80 fmol/mg protein). The ₁:₂-adrenoceptor ratio in both nonfailing atria was similar (about 70:30%) and was significantly smaller than in the different regions of both ventricles (about 80:20%). The ₁-subtype density was similar in nonfailing atria and ventricles (about 55 fmol/mg protein). The ₂-subtype density was significantly higher in the right and left atrium (about 25 fmol/mg protein) than in both ventricles (about 15 fmol/mg protein).In patients with end-stage heart failure due to idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy the total -adrenoceptor density was reduced by 50–60% in all regions. On the other hand, the ₁- and ₂-subtype distribution differed with the cause of heart failure. In patients with idiopathic dilated cardiomyopathy, the ₁-adrenoceptor density was lower in all regions, but the ₂-adrenoceptor density was not significantly reduced. In patients with ischaemic cardiomyopathy both ₁- and ₂-adrenoceptors were reduced in all regions.It is concluded that downregulation of -adrenoceptors in patients with end-stage idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy occurs uniformly throughout the heart. The results support the hypothesis that changes in -adrenoceptor subtypes may be related to the cause of heart failure.     

Chronic activation of the low affinity site of β1-adrenoceptors stimulates haemodynamics but exacerbates pressure-overload cardiac remodelling

BACKGROUND AND PURPOSE

The β₁-adrenoceptor has at least two binding sites, high and low affinity sites (β_1H and β_1L, respectively), which mediate cardiostimulation. While β_1H-adrenoceptor can be blocked by all clinically used β-blockers, β_1L-adrenoceptor is relatively resistant to blockade. Thus, chronic β_1L-adrenoceptor activation may mediate persistent cardiostimulation, despite the concurrent blockade of β_1H-adrenoceptors. Hence, it is important to determine the potential significance of β_1L-adrenoceptors in vivo, particularly in pathological situations.

EXPERIMENTAL APPROACH

C57Bl/6 male mice were used. Chronic (4 or 8 weeks) β_1L-adrenoceptor activation was achieved by treatment, via osmotic mini pumps, with (-)-{"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 (10 mg·kg⁻¹·day⁻¹). Cardiac function was assessed by echocardiography and micromanometry.

KEY RESULTS

(-)-{"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 treatment of healthy mice increased heart rate and left ventricular (LV) contractility. (-)-{"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 treatment of mice subjected to transverse aorta constriction (TAC), during weeks 4–8 or 4–12 after TAC, led to a positive inotropic effect and exacerbated fibrogenic signalling while cardiac hypertrophy tended to be more severe. (-)-{"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 treatment of mice with TAC also exacerbated the myocardial expression of hypertrophic, fibrogenic and inflammatory genes compared to untreated TAC mice. Washout of (-)-{"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 revealed a more pronounced cardiac dysfunction after 12 weeks of TAC.

CONCLUSIONS AND IMPLICATIONS

β_1L-adrenoceptor activation provides functional support to the heart, in both normal and pathological (pressure overload) situations. Sustained β_1L-adrenoceptor activation in the diseased heart exacerbates LV remodelling and therefore may promote disease progression from compensatory hypertrophy to heart failure.     

Evidence supporting the existence of presynaptic α-adrenoceptors in the regulation of endogenous noradrenaline release upon hepatic sympathetic nerve stimulation in the dog liver in vivo

Summary The existence and functional signficance of presynaptic -adrenoceptors within the liver was investigated in anesthetized dogs. The stimulation-evoked endogenous catecholamine overflow was determined in hepatic venous blood upon hepatic nerve stimulation (12 V, 1–8 Hz, 1 min). Under resting conditions, average plasma catecholamine levels in hepatic venous and aortic blood were 0.064 ng/ml and 0.334 ng/ml, respectively. A frequency-dependent increase (P<0.001) was found in the hepatic venous catecholamine overflow, while aortic catechlamine levels did not change significantly at any stimulation frequency tested. The increases in catecholamine overflow were associated with decreases in hepatic arterial vascular conductance (35–66%, P<0.001) at all frequencies tested. Yohimbine (0.3 mg/kg, i. v.) potentiated the catecholamine overflow by 110–140% (P<0.05) upon stimulation at low frequencies (1–4 Hz). Clonidine (20g/kg, i. v.) inhibited the catecholamine overflow by 55–76% (P<0.05) at frequencies of 1 and 2 Hz, and reduced the hepatic arterial response by 46% (P<0.01) at 1 Hz. The pretreatment with yohimbine (0.1 mg/kg, i. v.) abolished the inhibitory effects of clonidine both on the catecholamine overflow and the hepatic arterial responses. The results support the existence of a negative feedback mechanism mediated by presynaptic -adrenoceptors in the local regulation of noradrenaline release from hepatic sympathetic fibers. A functional significance of this process was suggested by an improved correlation found in the presence of clonidine between the catecholamine overflow and the hepatic arterial vascular conductance.     

Antagonism by γ-aminobutyric acid of the stimulant effect of angiotensin II on cardiac sympathetic ganglia in spinal dogs

Summary The effects of angiotensin II and neuro-aminoacids administered through the right subclavian artery (i. a.) to the cardiac sympathetic ganglia were investigated in spinal dogs. Angiotensin II (1–8 g) elicited a dose-dependent positive chronotropic effect which was reduced after i. a. injection of saralasin (100g). The effect of angiotensin II was not reduced after combined treatment with either hexamethonium (10 mg/kg) plus atropine (0.1 mg/kg) or hemicholinium-3 (5 mg/kg) plus preganglionic stimulation. The dosedependent response to angiotensin II of heart rate was inhibited by GABA (50, 500g), GABOB (500g) and muscimol (50, 100g). The inhibition of the response to angiotensin II by a small dose of GABA (50g), but not by a high one (500g), was antagonized by i. a. injection of picrotoxin (2 mg). The positive chronotropism induced by bethanechol (25, 50g) and a small dose of acetylcholine (25g) were significantly inhibited by a high dose (500g) but not by a low dose (50g) of GABA. These results confirm that angiotensin II stimulates cardiac chronotropism by acting on the angiotensin II receptor located at the cardiac ganglia and show that this stimulant effect is antagonized by GABA.     

A kinetic study of the release of noradrenaline by electrical stimulation: influence of presynaptic α-adrenoceptors

Summary Dog saphenous vein strips were incubated with 1.4 mol/l ³H-(-)-noradrenaline for 60 min, after inhibition of the noradrenaline-metabolizing enzymes and of extraneuronal uptake. At the end of the incubation period the strips were perifused for 150 min; cocaine (10 mol/l) was added to the perifusion fluid from t=75 min onwards. In some experiments either phentolamine (10 mol/l) or clonidine (0.1 mol/l) was also added at this time. Some strips were subjected to electrical stimulation from t=100 to 150 min of perifusion (t=0 being the start of perifusion), with frequencies ranging from 0.5 to 13.5 Hz. A compartmental analysis of spontaneous or electrically-induced efflux of ³H-noradrenaline was made. The spontaneous efflux had a long half time (t/2=124 min) and most of the ³H-noradrenaline which had accumulated in the strips did not participate in the efflux (bound fraction, representing 90% of tissue activity at t=100 min of perifusion). Neither phentolamine nor clonidine modified the half time or the bound fraction observed for spontaneous efflux. Electrical stimulation (>0.5 Hz) mobilized only one compartment of noradrenaline, which represented about 50% of the noradrenaline accumulated in the strips. The half time of ³H-efflux induced by electrical stimulation decreased when the frequency increased from 0.5 Hz up to 13.5 Hz. Phentolamine increased the rate of efflux for all frequencies of stimulation and decreased the half time of efflux. However, the releasable pool of noradrenaline was only increased by phentolamine at 0.5 Hz, but not at higher frequencies. Clonidine was used only at two frequencies of stimulation, 1.5 and 4.5 Hz. For the low frequency clonidine decreased the releasable pool, but no change was observed at 4.5 Hz.The results support the view that there is a norarenaline pool which is resistant to electrical stimulation and that its magnitude is not dependent on the activity of presynaptic -adrenoceptors.Results presented in part to the 13th Annual Meeting of the Portuguese Pharmacological Society (Porto, December 1982) and to the 5th Meeting on Adrenergic Mechanisms (Porto, October 1983)Work supported by a grant from Instituto Nacional de Investigação Científica (FmPl)     

Preponderance of β- over α-adrenoceptors in mediating the positive inotropic effect of phenylephrine in the ferret ventricular myocardium

Summary [³H]prazosin bound to the membrane fraction derived from the ferret ventricular muscle with high affinity in a saturable manner (K _d = 0.25 nmol/l and B _max = 27 fmol/mg protein in the right ventricle). [³H]CGP-12177, a -adrenoceptor ligand, bound to the membrane fraction with a _{K d} value of 0.29 nmol/l and a B _max of 42 fmol/mg protein. In the isolated ferret papillary muscle driven at 1 Hz at 37°C, phenylephrine elicited a concentration-dependent positive intropic effect. The maximal effect of phenylephrine was comparable to that of isoprenaline. Prazosin (0.3 ol/l) shifted the concentration-response curve for phenylephrine slightly but significantly to the right, the maximal response being unaffected. In contrast, bupranolol (0.3 gmol/l) shifted the curve for phenylephrine markedly downwards: the maximal response was depressed significantly to 40% and the curve became less steep. In the presence of prazosin and bupranolol the curve was shifted to the right, being essentially parallel to the control curve. These results indicate that in the ferret ventricular myocardium both - and -adrenoceptors mediate the positive inotropic effect of phenylephrine. The extent of contribution of the two classes of adrenoceptor is quite different from that in other mammalian species. In the ferret heart, -adrenoceptors predominate over -adrenoceptors in mediating the positive inotropic effect of phenylephrine, although the number of -adrenoceptors is not especially high when compared with other species. Send offprint requests to M. Endoh at the above address     

Facilitatory presynaptic angiotensin receptors on the sympathetic nerves of the human saphenous vein and pulmonary artery. Potential involvement in β-adrenoceptor-mediated facilitation of noradrenaline release

Summary Spirally cut strips of human saphenous vein and pulmonary artery preincubated with ³H-noradrenaline were superfused in the presence of corticosterone and desipramine or cocaine. In the saphenous vein angiotensin I, angiotensin II and angiotensin III concentration-dependently increased the electrically (2 Hz) evoked tritium overflow (relative order of potency: angiotensin II > angiotensin I > angiotensin III). The angiotensin receptor antagonist saralasin displaced the concentration-response curve of angiotensin II to the right, and also blocked the facilitatory effect of angiotensin III. Captopril, an inhibitor of angiotensin converting enzyme, did not modify the concentration-response curve of angiotensin I and did not significantly diminish the release-increasing effect of the nonselective -adrenoceptor agonist isoprenaline, whereas saralasin attenuated the facilitatory effect of the ₂-adrenoceptor agonist procaterol. In the pulmonary artery the angiotensin receptor agonist Val⁵-angiotensin II-Asp¹--amide also increased the electrically evoked tritium overflow in a concentration-dependent manner. It is concluded that the sympathetic nerve fibres of the human saphenous vein (and probably of the human pulmonary artery as well) are endowed with facilitatory presynaptic angiotensin receptors. Angiotensin I exerted its facilitatory effect in the saphenous vein probably via direct stimulation of angiotensin receptors but not by conversion to angiotensin II. Furthermore, the ₂-adrenoceptor-induced facilitation of noradrenaline release may in part be mediated by local stimulation of angiotensin II synthesis, which may occur by increased formation or activation of renin and/or increased availability of angiotensinogen.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert at the above address