Influence of the renin-angiotensin system on sympathetic neurotransmission in canine skeletal muscle in vivo

Summary The physiological importance of interactions between angiotensin II and sympathetic neurotransmission was studied in an in vivo model with constant flow blood perfused gracilis muscle in situ in dogs pretreated with desipramine and atropine. Sympathetic nerve stimulation- (2 and 8 Hz, 480 pulses) evoked overflow of endogenous noradrenaline and vasoconstriction, and vasoconstrictor responses to exogenous noradrenaline (0.5 nmol, locally i. a.) were evaluated.Angiotensin converting enzyme inhibition by benazeprilat (10 mg i. v.; n = 8) reduced arterial angiotensin II levels from 26 ± 8 to 2 +- 1 pM and reduced mean arterial and basal muscle perfusion pressures. Subsequent resubstitution of angiotensin II (3, 30 and 90 ng kg^–1 min^–1 i.v.) elevated arterial angiotensin II dose-dependently (to 67 ± 14, 622 ± 63 and 1940 ± 251 pM, respectively), as well as mean arterial and muscle perfusion pressures. Nerve stimulation-evoked noradrenaline overflow was unchanged following benazeprilat (–4 ± 4 and + 1 ± 8% at 2 and 8 Hz, respectively) and during subsequent infusions of angiotensin II. Vasoconstrictor responses to nerve stimulation and exogenous noradrenaline were also uninfluenced by these treatments. Thus, angiotensin II did not enhance sympathetic neurotransmission at the postjunctional level.Another group of animals was pretreated with noncompetitive -adrenoceptor blockade locally by phenoxybenzamine and benextramine (0.5 mg kg^–1 i. a. of each; n = 7), which abolished vasoconstrictor responses to nerve stimulation. The effects of benazeprilat and subsequent angiotensin II infusions (3 and 30 ng kg^–1 min^–1 i.v.) on circulating angiotensin II levels, mean arterial and muscle perfusion pressures were similar in this group. Following -adrenoceptor blockade, however, inhibition of angiotensin converting enzyme reduced sympathetic nerve stimulation-evoked noradrenaline overflow by 23 ± 4 and 21 ± 5% at 2 and 8 Hz, respectively (P < 0.01 for both). Angiotensin II infusions failed to enhance evoked noradrenaline overflow (–5 ± 10 and –18 ± 10% at 2 Hz; +6 ± 13% and –3 ± 14% at 8 Hz) also under these conditions.It is concluded that circulating angiotensin II does not influence sympathetic vascular control in canine skeletal muscle even at very high levels in arterial plasma. Angiotensin converting enzyme inhibition reduces nerve stimulation-evoked noradrenaline overflow only in the presence of -adrenoceptor blockade, suggesting that prejunctional -adrenoceptors have an overriding importance over prejunctional angiotensin II-receptors in the modulation of noradrenaline release in this model. The effect of converting enzyme inhibition may be related to merely local changes in angiotensin II concentration or — unrelated to the renin-angiotensin system — to other consequences of the blockade of this unspecific enzyme. Send offprint requests to J. Schwieler at the above address     

Potentiation of potassium-evoked noradrenaline and neuropeptide Y co-release by cardiac energy depletion: role of calcium channels and sodium-proton exchange

Summary The role of the cardiac energy status in the potassium-evoked exocytosis of both noradrenaline and the sympathetic co-transmitter neuropeptide Y (NPY) was investigated in the guinea-pig perfused heart. The transmitter release was stimulated by potassium depolarization (10–80 mmol/l) during normoxic perfusion (pO₂ > 100 mmHg) in the presence of glucose (11 mmol/l) and at various periods (5–40 min) of cardiac energy depletion. Energy depletion was induced either by anoxia (pO₂ < 5 mmHg) or by cyanide intoxication (1 mmol/l), both in combination with glucose-free perfusion. Endogenous noradrenaline and NPY were determined in the coronary venous overflow by high-pressure liquid chromatography combined with electrochemical detection and by radioimmunoassay, respectively.Under normoxic conditions potassium depolarization evoked a co-release of both transmitters [molar ratio 862 (noradrenaline) :1 (NPY)] at a threshold concentration of 40 mmol/l potassium. This transmitter overflow was characterized by its dependence on extracellular calcium and calcium influx through voltage-dependent neuronal calcium channels of the N-type. Cardiac energy depletion was accompanied by an acceleration and an enhancement of the potassium-evoked transmitter overflow. In comparison to normoxia, a 10-fold increased transmitter overflow with a comparable molar ratio [709 noradrenaline :1 (NPY)] was evoked by 40 mmol/l potassium after 10 min of either anoxia or cyanide intoxication. This sensitization to potassium depolarization reached a peak after 10 min of energy depletion and was characterized by a markedly reduced threshold concentration (10 mmol/l potassium). The enhanced sympathetic transmitter overflow in anoxia was suppressed by addition of glucose (11 mmol/l) to the perfusion buffer, suggesting that the sensitization of the overflow of noradrenaline and NPY to potassium depolarization requires a cessation of energy metabolism. The sensitization of the potassium-evoked (20 mmol/l) sympathetic transmitter overflow by energy depletion was further characterized: Consistent with an exocytotic release mechanism, the overflow was calcium-dependent. In contrast to normoxia, however, blockade of neuronal N-type calcium channels by either co-conotoxin (100 nmol/1) or cadmium chloride (50 mol/l) failed to reduce the potassium-evoked overflow of noradrenaline and NPY. In anoxia blockade of sodium-proton exchange by amiloride (1 mmol/l) or more specifically by ethylisopropylamiloride (1 mol/l) markedly attenuated the potassium-evoked transmitter overflow. Likewise, suppression of the potassium-evoked overflow of noradrenaline and NPY from the energy-depleted heart was achieved by extracellular acidosis (pH 6.0). In contrast, during normoxia blockade of sodium-proton exchange by either ethylisopropylamiloride (1 mol/l) or by extracellular acidosis (pH 6.0) did not affect the potassium-evoked (80 mmol/l) transmitter overflow. These findings suggest that the sensitization of sympathetic nerve endings to potassium depolarization, caused by cardiac energy depletion, requires sodium entry into the sympathetic nerve ending via sodium-proton exchange.The results of the present study indicate, that the threshold concentration for the potassium-evoked exocytotic release of noradrenaline and NPY from the guinea-pig isolated perfused heart is intimately coupled to the energy status of cardiac sympathetic nerve fibres. The energy status not only determines the quantity of the transmitters released but also the mode of sodium and calcium entry triggering the depolarization-evoked transmitter overflow.Preliminary findings were reported at the 63rd Scientific Sessions of the American Heart Association, Dallas/USA (Haass et al., 1990b) and at the Annual Meeting of the European Section of the International Society for Heart Research, Leuwen/Belgium (Haass et al. 1991b)Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion and ihre Regulation) Correspondence to M. Haass at the above address     

Peripheral presynaptic and central effects of clonidine,yohimbine and rauwolscine on the sympathetic nervous system in rabbits

Summary The function of presynaptic ₂-autoreceptors at postganglionic sympathetic neurones under conditions of normal, ongoing sympathetic impulse traffic was studied in anaesthetized rabbits (alfadolone + alfaxalone). Clonidine was used as an ₂-adrenoceptor agonist, and yohimbine and rauwolscine were used as antagonists. Mean arterial pressure, postganglionic renal sympathetic firing rate, arterial plasma noradrenaline concentration and heart rate were measured before (basal values) and at the end of 3-min infusions of sodium nitroprusside and phenylephrine, which were given to modulate efferent activity in the sympathetic nervous system through the baroreflex.The nitroprusside- and phenylephrine-induced changes of mean arterial pressure produced the expected changes in sympathetic nerve activity, plasma noradrenaline and heart rate. Clonidine (5 µg kg^–1 + 0.5 µg kg^–1 min^–1) reduced the basal mean arterial pressure, sympathetic nerve activity and heart rate. It also reduced the nitroprusside-induced increase in the plasma noradrenaline level without changing the nitroprusside-induced increase in sympathetic firing. These results, as well as the mean arterial pressure-sympathetic nerve activity and the sympathetic nerve activity-plasma noradrenaline function curves indicate that clonidine inhibited both sympathetic tone centrally and the average release of noradrenaline per action potential peripherally. Yohimbine (1 mg kg^–1 + 0.1 mg kg^–1 h^–1) and rauwolscine (0.5 mg kg^–1 + 0.1 mg kg^–1 h^–1) increased the basal plasma noradrenaline level without any increase of renal sympathetic nerve activity. They also enhanced the nitroprusside-induced increase in plasma noradrenaline without any enhancement of the nitroprusside-induced increase in sympathetic firing. The hypotensive response to nitroprusside was attenuated, whereas the heart rate response was augmented. These results, as well as the mean arterial pressure-sympathetic nerve activity and the sympathetic nerve activity-plasma noradrenaline function curves indicate that the main effect of yohimbine and rauwolscine was to increase the average release of noradrenaline per action potential.The simultaneous measurement of postganglionic sympathetic nerve activity and the arterial plasma noradrenaline concentration proved suitable to differentiate central (or ganglionic; this distinction was not possible) effects of ₂-adrenoceptor ligands from peripheral presynaptic effects. The results show that endogenous presynaptic, ₂-adrenergic autoinhibition of noradrenaline release from postganglionic sympathetic neurones operates physiologically in anaesthetized rabbits with ongoing, uninterrupted sympathetic nerve activity. The results also indicate that blockade of ₂-autoreceptors enhances the sympathetic reflex compensatory response to a hypotensive stimulus. Send offprint requests to B. Szabo at the above address     

Phorbol ester,an activator of protein kinase C,enhances calcium-dependent release of sympathetic neurotransmitter

Summary The effect of phorbol ester, phorbol 12,13-dibutyrate, was investigated on the overflow of tritium from ³H-noradrenaline-loaded sympathetic neurons of the isolated perfused salivary gland of the rat. Stimulation (1 Hz for 60 s)-evoked overflow of tritium was enhanced by phorbol ester. A significant enhancement was seen at 1 nmol/l, which increased to a maximum level (over 4-fold) at 30 nmol/l. The spontaneous overflow of radioactivity, however, was not affected by any concentration of phorbol ester. The facilitatory effect of phorbol ester on stimulation-evoked overflow was observed in the presence of inhibitors of neuronal and extraneuronal uptake as well as after removal of negative feedback inhibition of release by presynaptic alpha-adrenoceptors. Tyramine (7 mol/l for 10 min) caused a marked increase in the overflow of tritium in either the presence or absence of calcium. However, tyramine-induced overflow was not enhanced by phorbol ester. It is concluded that protein kinase C of sympathetic neurons is involved in an exocytotic release of the transmitter.     

Nicotine-induced release of noradrenaline and neuropeptide Y in guinea-pig heart: role of calcium channels and protein kinase C

Summary The role of calcium, calcium influx through calcium channels, and activation of protein kinase C for the nicotine-induced release of noradrenaline and of the sympathetic co-transmitter neuropeptide Y (NPY) was investigated in the guinea-pig isolated perfused heart. In the coronary venous overflow noradrenaline and NPY were determined by high-pressure liquid chromatography and radioimmunoassay, respectively. In the presence of extracellular calcium (1.85 mmol/l) nicotine (1–100 mol/l) evoked a concentration-dependent overflow of both transmitters with a molar ratio of approximately 1500 (noradrenaline):1 (NPY). The nicotine-induced (100 mol/l) overflow of noradrenaline and NPY was in a linear manner related (r = 0.79 and 0.90, respectively; p < 0.05) to the extracellular calcium concentration (0–1.85 mmol/l), and it was prevented by calcium-free perfusion. The L-type calcium channel blocker felodipine (100 nmol/l) did not affect the nicotine-induced (100 mol/l) transmitter overflow. On the other hand, the neuronal (N-type) calcium channel blockers -conotoxin (100 nmol/l) and cadmium chloride (50 mol/l) reduced the nicotine-induced (100 pmol/l) transmitter overflow to 20% of the control value, suggesting a role of N-type calcium channels in mediating the calcium influx for the nicotine-induced transmitter release. The nicotine-induced (30 mol/l) overflow of both transmitters was two- to three-fold increased by activation of protein kinase C (phorbol 12-myristate 13-acetate; 100 nmol/l). The transmitter overflow was unaffected by 4-phorbol 12,13-didecanoate (100 nmol/l), a phorbol ester which does not stimulate protein kinase C. Further supporting a modulatory role of protein kinase C, inhibition of the enzyme by either polymyxin B (100 gmol/I) or by cremophor RH-30 (1mol/l) almost completely suppressed the overflow of noradrenaline and NPY. The results of the present study indicate that nicotine evokes a concentration-dependent exocytotic co-release of noradrenaline and NPY in the guinea-pig isolated perfused heart which is characterized by its dependence on extracellular calcium, calcium influx through N-type calcium channels and activation of protein kinase C.This work was supported by a grant from the Forschungsrat Rauchen und Gesundheit Send of fprint requests to M. Haass at the above address     

Characterization and presynaptic modulation of stimulation-evoked exocytotic co-release of noradrenaline and neuropeptide Y in guinea pig heart

Summary The relationship between noradrenaline and neuropeptide Y (NPY) release was investigated in the in situ perfused guinea pig heart with intact sympathetic innervation. For determination of NPY concentrations in the perfusate, a specific radioimmunoassay was employed and further characterized. Electrical stimulation of the left stellate ganglion (4, 8, 12, and 50 Hz; for 10 min) evoked a calcium-dependent and frequency-related overflow of noradrenaline and NPY, which was positively correlated (r = 0.83; p < 0.001; n = 25). When two subsequent stimulations (12 Hz; each for 1 min) were performed in the same heart, addition of noradrenaline (10 M) 5 min prior to the second stimulation reduced NPY overflow by 43 ± 10%. The stimulated release of noradrenaline and NPY was increased by the alpha₂-adrenoceptor antagonist yohimbine (1 M) to 170 ± 10% and 199 ± 26%, and attenuated by the alpha₂-adrenoceptor agonist B-HT 920 (1 M) to 70 ± 9% and 68 ± 9%, respectively. The adenosine analogue cyclohexyladenosine (1 M) significantly reduced the stimulated overflow of both noradrenaline (to 57 ± 5%) and NPY (to 73 ± 8%). Exogenous NPY (100 nM) attenuated the stimulated overflow of noradrenaline by 30 ± 6%. Uptake₁ blockade with desipramine (100 nM) or nisoxetine (100 nM) prior to the second stimulation significantly increased noradrenaline overflow and attenuated that of NPY; the attenuation of the stimulation-evoked overflow of NPY was abolished by yohimbine (1 M).Our results indicate that electrical stimulation induces a calcium-dependent, exocytotic co-release of noradrenaline and NPY. The co-release of both transmitters is regulated by presynaptic receptors in a parallel manner; furthermore, both transmitters, noradrenaline and possibly NPY, modulate their own release by a presynaptic negative feedback mechanism via presynaptic alpha₂-adrenoceptors and NPY-receptors.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 320 - Herzfunktion und ihre Regulation). Presented in part at the 61st Scientific Sessions of the American Heart Association. Washington DC, November 1988 Send offprint requests to M. Haass at the above address     

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.     

A presynaptic excitatory M1 muscarine receptor at postganglionic cardiac noradrenergic nerve fibres that is activated by endogenous acetylcholine

Summary Rabbit atria were isolated with the extrinsic right vagus and sympathetic nerves intact and perfused with Tyrode solution. Noradrenaline overflow evoked by sympathetic nerve stimulation (SNS) at 3 Hz for 3 min was determined before, during, and after vagus nerve stimulation (VNS), also at 3 Hz and for 3 min. The VNS pulses preceded the SNS pulses by 3, 100 and 233 ms. Acetylcholine overflow was determined after labelling of the transmitter stores with [¹⁴C]choline.Pirenzepine 80 nmol/l failed to alter the muscarinic inhibition of noradrenaline overflow when the vago-sympathetic impulse intervals were 3 and 233 ms. At an interval of 100 ms VNS did not significantly inhibit noradrenaline overflow in the absence of pirenzepine but produced an inhibition in the presence of the drug. When the pirenzepine concentration was varied (0.4–300 nmol/l) the largest inhibition of noradrenaline overflow was observed at 5.7 nmol/l whereas 300 nmol/l fully antagonized the inhibition. Acetylcholine overflow evoked by VNS was not altered by pirenzepine 0.4–300 nmol/l.AF-DX 116 (11-[{2[oi(diethylamino)methyl]-1-piperidinyl}-acetyl]-5,11-dihydro-6H-pyrido-[2,3-b]-[1,4]benzodiazepine-6-one), an M₂ receptor selective antagonist, concentration-dependently (100–800 nmol/l) inhibited the decrease of tension development elicited by VNS. At the 100 ms vago-sympathetic impulse interval noradrenaline overflow was enhanced in the presence of AF-DX 116 400 and 800 nmol/l. However, already 100 nmol/l of the drug caused a maximum (fourfold) increase of acetylcholine overflow.It is concluded that acetylcholine released onto noradrenergic nerve fibres causes a small facilitation of noradrenaline overflow at a vago-sympathetic impulse interval of 100 ms. This response is mediated by an M₁ receptor and is superimposed on the well-known M₂ receptor mediated inhibition of noradrenaline release which is obtained at vago-sympathetic impulse intervals ranging between 3 and 233 ms. The M₂ autoreceptor regulating acetylcholine release is activated by lower synaptic concentrations of the transmitter than the M₂ heteroreceptor regulating noradrenaline release.Abbreviations SNS sympathetic nerve stimulation - VNS vagus nerve stimulation Send offprint requests to: E. Muscholl at the above address     

Role of angiotensin and sodium intake in cardiac noradrenaline release

Summary The effects of exogenous and of endogenous angiotensin on noradrenaline overflow were investigated in saline perfused rat hearts with intact sympathetic nerves. The overflow of endogenous noradrenaline induced by electrical stimulation of the left stellate ganglion was determined in the coronary venous effluent by HPLC. The activity of the renin-angiotensin system was modulated by varying of the nutritional sodium load prior to the experiments. Endogenous angiotensin formation was blocked by angiotensin-converting enzyme inhibitors.Following high sodium intake, both angiotensin II (100 nmol/l) and angiotensin I (1 µmol/l) caused a marked increase of the electrically evoked noradrenaline overflow. After inhibition of the angiotensin-converting enzyme using captopril (350 nmol/l) or ramiprilat (50 nmol/l), angiotensin I (1 µmol/l) did not enhance noradrenaline overflow. This indicates an active cardiac angiotensin conversion, since the sole administration of captopril and ramiprilat did not affect noradrenaline overflow in rats with high sodium intake. Following low sodium intake, neither angiotensin II (100 nmol/l) nor angiotensin I (1 µmol/l) significantly affected noradrenaline overflow. Both captopril and ramiprilat, however, significantly reduced noradrenaline overflow induced by electrical stimulation, suggesting a facilitory action of endogenous angiotensin under these conditions.This concept was substantiated when evaluating the noradrenaline overflow during control stimulations. Following low sodium intake, stimulation evoked noradrenaline overflow was higher as compared to that after nutritional sodium load. The results are in keeping with a sodium-dependent intracardiac formation of angiotensin II which facilitates noradrenaline release from sympathetic nerve terminals. Following low sodium intake, cardiac angiotensin II formation is active, as indicated by the suppression of noradrenaline release by angiotensin-converting enzyme inhibitors and the ineffectiveness of exogenous application of angiotensin II. In contrast, suppression of endogenous angiotensin 11 formation in sodium loaded animals sensitizes the sympathetic nerves to exogenous angiotensin.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion and ihre Regulation)Presented in part at the 63nd Scientific Sessions of the American Heart Association, Dallas/USA, November 1990 Send offprint requests to G. Richardt at the above address     

Neuropeptide Y differentiates between exocytotic and nonexocytotic noradrenaline release in guinea-pig heart

Summary The overflow of neuropeptide Y (NPY; radioimmunoassay), noradrenaline and dihydroxyphenylethylenglycol (DOPEG; high pressure liquid chromatography) from guinea-pig perfused hearts was investigated in relationship to exocytotic and nonexocytotic release mechanisms. Exocytotic release: Electrical stimulation of the left stellate ganglion (12 Hz; 1 min) evoked a calcium-dependent overflow of noradrenaline and NPY, that was accompanied by a minor and prolonged increase in DOPEG overflow. This increase in DOPEG overflow was attenuated by blockade of neuronal amine re-uptake. In the presence of calcium, a closely related co-release of noradrenaline and NPY was also observed during administration of veratridine (10 M); it was completely prevented by tetrodotoxin (1 M). Nonexocytotic release: In the absence of extracellular calcium, veratridine (30 M) induced noradrenaline overflow only when combined with the reserpine-like agent Ro 4-1284 (10 M). This overflow was accompanied by efflux of DOPEG, but not of NPY. Similarily, tyramine (1–100 M) induced a calcium-independent concomitant overflow of both noradrenaline and DOPEG, but not of NPY. During anoxic and glucose-free perfusion a predominantly calcium-independent overflow of noradrenaline was observed; only in the presence of extracellular calcium was this overflow accompanied by a minor overflow of NPY. Noradrenaline overflow, induced by veratridine plus Ro 4-1284 (in the absence of calcium), by tyramine, or by anoxia, was suppressed by blockade of neuronal amine re-uptake, and was, therefore, mediated by reversed transmembrane amine transport by the neuronal uptake₁ carrier.The results indicate that NPY is co-released with noradrenaline only during calcium-dependent exocytosis. On the other hand, whenever, noradrenaline is released by non-exocytotic (calcium-independent and carrier-mediated) release mechanisms, no substantial NPY overflow is observed. The simultaneous determination of noradrenaline and NPY overflow, therefore, allows a differentiation between exocytotic and nonexocytotic noradrenaline release, and NPY may be utilized as a marker of exocytotic noradrenaline release.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion und ihre Regulation)Presented in part at the 62nd Scintific Sessions of the American Heart Association, New Orleans/USA, November 1989     

Nicotine receptors do not modulate the 3H-Noradrenaline release from the isolated rat heart evoked by sympathetic nerve stimulation

Summary Isolated rat hearts with the right sympathetic nerves attached were perfused at a constant flow rate of 7 ml/min with Tyrode's solution. (-)-³H-Noradrenaline (final concentration 10–13.9 nM) was infused for 10 min to label the noradrenaline stores. After wash-out the sympathetic nerves were stimulated electrically (3 Hz, 180 impulses, 1 ms, 20–30 mA) three times (S1–S3) at intervals of 15 min. ³H-Noradrenaline and its metabolites were determined by liquid scintillation counting according to Graefe et al. (1973).Both, nicotine 50 M and p-aminophenethyltrimethylammonium (PAPETA) 30 M, enhanced the ³H-noradrenaline overflow in the absence of nerve stimulation. The effect of PAPETA was biphasic and was still observed in the presence of N-methylatropine 0.1 M. Hexamethonium 10 M abolished the first phase only, but cocaine 10 M antagonized both phases.The decline of the stimulation-evoked overflow of ³H-noradrenaline from the first to the third stimulation period was similar in the absence and in the presence of cocaine 10 M starting before S1 and perfused throughout. Cocaine 10 M added before S2, however, enhanced the evoked overflow by 77%.PAPETA 30 M increased the stimulation-evoked overflow by 67% in the absence, and by 73% of the respective control in the presence, of hexamethonium 10 M. PAPETA 30 M failed to enhance the evoked overflow in the presence of cocaine. Hexamethonium (added before S2) did not modify the effectiveness of nerve stimulation.Nicotine, neither when perfused from 6 min before S2, nor when added to the perfusion fluid simultaneously with the onset of nerve stimulation, caused changes in the ³H-noradrenaline output upon S2.Upon stimulation a rather discrete increase in ³H-DOPEG overflow was observed. This increase was abolished by cocaine and/or PAPETA.It is concluded that nicotine and PAPETA stimulate the output of ³H-noradrenaline from the rat heart sympathetic nerves by activation of nicotine receptors. However, the amount of transmitter released is small. Neither drug appeared to modulate the output of ³H-noradrenaline upon electrical nerve stimulation via nicotine receptors.PAPETA, like cocaine, appears to block the reuptake of released transmittsrs thereby enhancing the ³H-noradrenaline overflow and reducing the overflow of ³H-DOPEG (formed intraneuronally from recaptured noradrenaline after nerve stimulation).Abbreviations used DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MOPEG 3-methoxy-4-hydroxy-phenylglycol - NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites (sum of MOPEG and VMA) - PAPETA p-aminophenethyltrimethylammonium - VMA 3-methoxy-4-hydroxymandelic acid     

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.     

Interaction between presynaptic facilitatory angiotensin II receptors and inhibitory muscarinic cholinoceptors on3H-noradrenaline release in the rabbit heart

Summary The existence of a functional interaction between presynaptic receptors modulating the release of noradrenaline was studied in the rabbit heart. Isolated right atria were prelabelled with³H-noradrenaline and the overflow of tritium was induced by field stimulation (2 Hz, 0.1 ms duration, supramaximal voltage for a total of 180 pulses). In atria superfused with Krebs' solution containing 10 mol/l cocaine and 30 mol/l corticosterone, angiotensin II (10 nmol/l) increased the stimulation-evoked overflow of³H-transmitter by 2.8-fold. The addition of atropine (0.3 mol/l) to the perfusion medium, either in the presence or in the absence of uptake inhibitors, further enhanced the facilitatory effect of angiotension II (³H-transmitter release increased by 3.5-fold). Exposure to 1 mol/l carbachol decreased by 65% the stimulation-evoked release of³H-transmitter while the facilitatory effect of angiotensin II determined in the presence of the muscarinic cholinoceptor agonist was enhanced (³H-transmitter release increased by 6.6-fold). Conversely, during sustained activation of presynaptic angiotensin receptors producing a 2.5-fold increase in the release of³H-transmitter, the inhibitory effect of carbachol remained unchanged. These results suggest a functional interaction between presynaptic inhibitory muscarinic cholinoceptors and the presynaptic facilitatory angiotensin receptor which modulate the release of noradrenaline from cardiac noradrenergic nerves.     

Noradrenaline release from slices of the thalamus of normal and morphine-dependent rats

Summary The effect of morphine on potassium-induced stimulation of (³H)-noradrenaline release from slices of the rat thalamus was investigated. The in vitro addition of morphine (10^–6 M) significantly depressed potassium-induced tritium overflow by 42% and this was prevented by the prior addition of naloxone (3×10^–6 M) to the medium. The stimulation-evoked overflow of tritium from slices of the thalamus of morphine-dependent rats was not significantly different from normal controls. Addition of naloxone (10^–5 M) 10 min before exposure of the tissues to 20 mM K⁺ significantly enhanced noradrenaline release from dependent slices. The results suggest that the basic release mechanism may have adapted to the continuous presynaptic inhibition of release by morphine.     

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.     

Effect of phentolamine on noradrenaline uptake and release

Summary The influence of phentolamine on the uptake of exogenous noradrenaline infused into the aortic cannula and on the overflow of endogenous noradrenaline caused by sympathetic nerve stimulation was investigated in the isolated perfused rabbit heart. 10^–6 M phentolamine doubled the overflow of endogenous noradrenaline, but did not change noradrenaline uptake. 10^–5 M phentolamine increased the stimulation-induced overflow of noradrenaline 4-fold and inhibited amine uptake by about 50%. 10^–4 M phentolamine elevated the overflow of noradrenaline less than 10^–5 and 3×10^–5 M did. The augmentation of transmitter overflow was only partly reversed by 13 min perfusion with drug-free medium.Pretreatment of hearts with 1.5×10^–5 M cocaine or with 10^–7 or 10^–6 M desipramine did not change the effect of phentolamine on the overflow of noradrenaline evoked by nerve impulses. Pretreatment of hearts with 10^–5 M, but not with 10^–6 M, phentolamine prevented the increase of transmitter overflow by cocaine.It is concluded that low concentrations of phentolamine potentiate the overflow of noradrenaline during nerve stimulation by a mechanism different from that of cocaine, i.e. different from inhibition of neuronal re-uptake. The nature of this mechanism is discussed.This work was supported by the Deutsche Forsehungsgemeinschaft. We have the pleasure to thank Mrs. Ch. Arts, Miss B. Piel and Mr. E. Hagelskamp for skilful technical assistance.     

Autoinhibition of noradrenaline release from the rat heart as a function of the biophase concentration. Effects of exogenous α-adrenoceptor agonists,cocaine, and perfusion rate

Rat isolated perfused hearts with the right sympathetic nerves intact were loaded with 3H-(-)-noradrenaline. The nerves were stimulated with trains of 180 pulses at 3 Hz and at 10 min intervals. The overflow of 3H-noradrenaline and 3H-metabolites was determined by liquid scintillation spectrometry. Clonidine (IC50 17 nM), oxymetazoline (IC50 63 nM), and alpha-methylnoradrenaline (apparent IC50 35 nM, determined in the presence of cocaine and propranolol) decreased the stimulation-evoked overflow of 3H-noradrenaline by 26, 49, and 78%, respectively, but not methoxamine up to 100 microM (propranolol present). Oxymetazoline and alpha-methyl-noradrenaline did not cause desensitization of the presynaptic adrenoceptors when present at their IC80 for 33 min. At a perfusion rate of 7 ml/min, yohimbine 1 microM enhanced the stimulation-evoked 3H-noradrenaline overflow by 26% in the absence, and by 58% in the presence of cocaine. Phentolamine 1 microM increased it by 69% when the neuronal reuptake was blocked. The increase by the antagonists faded with successive period of nerve stimulations, and was positively correlated with the biophase concentration of noradrenaline as reflected by the amount of 3H-noradrenaline released into the perfusate per nerve stimulation. At a perfusion rate of 1.8 ml/min (neuronal reuptake blocked), yohimbine 1 microM increased the overflow by 127%. The results indicate that the alpha 2-adrenoceptor-mediated autoinhibition in the rat perfused heart depends on the clearance of transmitter from the biophase via neuronal reuptake and diffusion into the vascular space. Reduction of either elimination pathway enhances the biophase concentration of noradrenaline, thus increasing the autoinhibition of release.     

Presynaptic inhibitory opioid delta- and kappa-receptors in a branch of the rabbit ileocolic artery

The largest rami caecales of the ileocolic artery (a branch of the mesenteric artery) were perfused at a constant rate of flow. Either vasoconstriction or the release of previously incorporated [3H]noradrenaline was measured. The following opioid agonists inhibited the vasoconstriction elicited by electrical field pulses (5 pulses at 10 Hz; EC50 values in brackets): [Leu5]enkephalin (596 nmol/l), [D-Ala2,D-Leu5]enkephalin (69 nmol/l), dynorphin-(1-13) (366 nmol/l) and ethylketocyclazocine (668 nmol/l). Fentanyl (up to 30 mumol/l) and normorphine (up to 100 mumol/l) caused at best minimal inhibition. The effects of [Leu5]enkephalin and dynorphin-(1-13) were antagonized by naloxone. Only the effect of [Leu5]enkephalin but not that of dynorphin-(1-13) was antagonized by the delta-selective antagonist ICI 154129. [Leu5]Enkephalin and dynorphin-(1-13) did not decrease the vasoconstrictor response to exogenous noradrenaline or ATP. In arteries preincubated with [3H]noradrenaline, electrical stimulation (50 pulses at 1 Hz) increased the outflow of tritium. The stimulation-evoked overflow was reduced by [Leu5]enkephalin and dynorphin-(1-13), and the effect of [Leu5]enkephalin was antagonized by naloxone. It is concluded that the postganglionic sympathetic neurons of the ramus caecalis possess presynaptic opioid receptors which, when activated, inhibited transmitter release. The receptors appear to be of the delta- and kappa- but not the mu-type.     

Effects of the neuropeptide Y Y2 receptor antagonist BIIE0246 on sympathetic transmitter release in the pig in vivo

This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y(2) receptor antagonist, on renal sympathetic nerve-evoked release of noradrenaline and NPY-like immunoreactivity (-LI) in the pig in vivo.Stimulation (5 Hz, 60 s) of renal sympathetic nerves evoked vasoconstriction and overflow of noradrenaline and NPY-LI. Infusion of peptide YY (PYY; 1 microg/kg per min) strongly inhibited the stimulation-evoked overflow of noradrenaline and NPY-LI. BIIE0246 (100 nmol/kg) abolished the inhibitory effect of PYY. BIIE0246 did not however affect the stimulation-evoked overflow of noradrenaline and NPY-LI, or basal cardiovascular parameters, per se. The alpha(2)-adrenoceptor antagonist yohimbine (0.2 mg/kg) significantly elevated both basal plasma levels and the nerve stimulation-evoked overflow of noradrenaline and NPY-LI. Subsequent administration of BIIE0246 did not further alter these effects. However, BIIE0246 caused splenic vasodilatation per se when given after yohimbine.It is concluded that the renal sympathetic nerves in the pig possess NPY Y(2) receptors, which upon activation inhibit transmitter (noradrenaline and NPY) release. Furthermore, when circulating levels of NPY were enhanced after blockade of alpha(2)-adrenoceptors, an involvement of endogenous NPY, acting on the NPY Y(2) receptor, in regulation of basal splenic vascular tone was unveiled.     

Search for an endogenous cannabinoid-mediated effect in the sympathetic nervous system

Activation of CB₁ cannabinoid receptors by exogenous agonists causes presynaptic inhibition of neurotransmitter release from axon terminals. In the central nervous system, presynaptic CB₁ receptors can also be activated by endogenous cannabinoids (endocannabinoids) released from postsynaptic neurons. Except in the vas deferens, there is no indication of endocannabinoid-mediated presynaptic inhibition in the sympathetic nervous system. The aim of the present study was to search for such inhibition in pithed rats. Artificial sympathetic tone was established by continuous electrical stimulation of preganglionic sympathetic axons. The CB₁ cannabinoid receptor antagonist rimonabant (0.5 and 2 mg kg^–1 i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration. Since activation of G_q/11 protein-coupled receptors enhances endocannabinoid synthesis in the central nervous system, we attempted to stimulate endocannabinoid production by infusion of arginine vasopressin and phenylephrine (both activate G_q/11 protein-coupled receptors). Rimonabant (2 mg kg^–1 i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration during infusion of phenylephrine or vasopressin. In the final series of experiments we verified that an exogenous cannabinoid agonist produces sympathoinhibition. The synthetic CB₁/CB₂ receptor agonist WIN55212-2 (0.1 and 1 mg kg^–1 i.v.) markedly lowered blood pressure and plasma noradrenaline concentration in pithed rats with electrically stimulated sympathetic outflow. In contrast, in pithed rats with a pressor infusion of noradrenaline, WIN55212-2 did not change blood pressure or heart rate. The results verify that activation of peripheral presynaptic CB₁ receptors inhibits noradrenaline release from sympathetic nerve terminals. The lack of effect of the CB₁ receptor antagonist rimonabant indicates that, even under conditions favouring endocannabinoid synthesis, endocannabinoid-mediated presynaptic inhibition is not operating in the sympathetic nervous system of the pithed rat.