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     

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     

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     

Depression by neuropeptide Y of noradrenergic inhibitory postsynaptic potentials of locus coeruleus neurones

Summary Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus (LC). The spontaneous firing of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. Focal electrical stimulation evoked a synaptic depolarization (PSP) followed by a hyperpolarization (IPSP). Neuropeptide Y (NPY; 0.1 mol/l) inhibited the IPSP only. Pressure ejection of noradrenaline produced hyperpolarization which was potentiated in the presence of NPY (0.1 mol/l). Hence, NPY appears to inhibit the release of noradrenaline from dendrites or recurrent axon collaterals of LC neurones. Correspondence to: P. Illes at the above address     

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     

Inhibition of noradrenaline release by neuropeptide Y in mouse atria does not involve inhibition of adenylate cyclase or a pertussis toxin-susceptible G protein

Summary Neuropeptide Y (30–1000 nmol/1) significantly inhibited the fractional stimulation-induced outflow of radioactivity from mouse atria preincubated with [³H-noradrenaline. The inhibitory effect of neuropeptide Y was observed at all frequencies tested (2, 5 and 10 Hz) as well as after a-adrenoceptor blockade with phentolamine (1 µmol/l). A combination of 8-bromo adenosine cyclic-3-5-mono-phosphate (90 or 270 µmol/l) with the phosphodiesterase inhibitor 3-isobutyl-I-methylxanthine (100 µmol/l) was used to saturate maximally the adenylate cyclase system and these drug combinations significantly enhanced the stimulation-induced outflow of radioactivity. However, neuropeptide Y inhibited the stimulation-induced outflow in the presence of these drugs, suggesting that the inhibitory effect of neuropeptide Y was not due to decreasing endogenous cyclic AMP formation. Finally, atria from mice treated with pertussis toxin were used. In this case, the inhibitory effect of neuropeptide Y on the stimulation-induced outflow of radioactivity was still observed suggesting that inhibitory prejunctional neuropeptide Y receptors are not coupled to a pertussis toxin-susceptible G protein. Send offprint requests to Sylvain Foucart at the above address     

Increased noradrenaline release in rat portal vein during sympathetic nerve stimulation due to activation of presynaptic beta-adrenoceptors by noradrenaline and adrenaline.

With the aim of investigating whether exogenous noradrenaline (NA) and adrenaline (A) can modulate transmitter release via the stimulation of presynaptic beta-adrenoceptors, 3H-release from isolated portal veins was studied after pretreatment with 3H-1-NA, phenoxybenzamine, desipramine and normetanephrine. NA (10 muM) and A (0.05 muM) increased the fractional 3H-release elicited by sympathetic nerve stimulation by 30%. This effect could be blocked by d, 1-propranolol which per se reduced the release by 10%. It is concluded that NA can facilitate its own release via a presynaptic beta-adrenoceptor-mediated positive feed-back mechanism and that adrenaline can stimulate this beta-adrenoceptor-mediated mechanism.     

Effects of noradrenaline and neuropeptide Y on rat mesenteric microvessel contraction

We have studied the contractile effects of the sympathetic transmitter noradrenaline and its cotransmitter neuropeptide Y (NPY) given alone and in combination on isolated rat mesenteric resistance vessels (200–300 m diameter). Noradrenaline and NPY each concentration-dependently contracted rat mesenteric microvessels (EC₅₀ 800 nM and 10 nM, respectively), but noradrenaline caused considerably greater maximal effects than NPY (14.3 mN vs. 3.5mN). A low antagonistic potency of yohimbine indicated that the response to noradrenaline did not involve ₂-adrenoceptors, and the subtype-selective antagonists 5-methylurapidil, tamsulosin and chloroethylclonidine indicated mediation via an _1A-adrenoceptor. Shallow Schild regressions for prazosin and 5-methylurapidil indicated that an ₁-adrenoceptor subtype with relatively low prazosin affinity might additionally be involved. Studies with the NPY analogues PYY, [Leu³¹, Pro³⁴]NPY and NPY_18–36 demonstrated that NPY acted via a Y₁ NPY receptor. In addition to its direct vasoconstricting effects NPY also lowered the noradrenaline EC₅₀ but did not appreciably affect maximal noradrenaline responses indicating possible potentiation. The potentiating NPY response occured with similar agonist potency as the direct contractile NPY effects and also via a Y₁ NPY receptor. The Ca²⁺ entry blocker nitrendipine (300 nM) reduced direct contractile responses to noradrenaline and NPY but did not affect the potentiation response to NPY.     

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     

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.     

Prednisolone-3,20-bisguanylhydrazone: binding in vitro to sodium-and-potassium-activated adenosine triphosphatase of guinea pig heart ventricular muscle.

Spontaneously hypertensive rats and normotensive Kyoto Wistar controls were divided into 3 groups of 10 animals each and treated with phenoxybenzamine (5 mg/kg once daily), propranolol (25 mg/kg twice daily) or saline (once daily). After 5 weeks the in vitro incorporation of D-[U-14C]-glucose into aortic lipids and glycogen was measured in the presence and absence of insulin (1 mU/ml). In both normotensive and hypertensive rats treated with propranolol 14C-incorporation into triglycerides was reduced. Furthermore, insulin significantly stimulated 14C-incorporation into triglycerides, phospholipids and glycogen in propranolol-treated hypertensive rats. This effect was not statistically significant (0.05 less than p less than 0.1) in propranolol-treated normotensives. Phenoxybenzamine treatment did not significantly modify aortic lipogenesis or glycogen synthesis from glucose. Chronic propranolol treatment of spontaneously hypertensive rats resulted in aortic tissue becoming sensitized to insulin. Possible mechanisms and explanations for this are discussed.     

Multiple electrophysiological actions of amiodarone on guinea pig heart

Summary The cellular electrophysiologic effects of acute exposure to amiodarone (AM) on guinea pig papillary muscle (PM) and Purkinje fibres (PF) were investigated by means of conventional microelectrode techniques. Superfusion with AM < 1.1 × 10^–4 mol/l reduced the maximum rate of rise ( _max) of the action potential (AP) upstroke phase 0 of both PM and PF stimulated at 1 Hz, without changing resting membrane potential (RMP) or action potential duration (APD). AM > 1.1 × 10^–4 mol/l decreased APD at all levels, accompanied by decreases in _max and RMP. PF AP's were much more sensitive to AM than PM. In contrast, chronic exposure (20 mg/kg/day, 3/12 weeks) prolonged PM APD at all levels and decreased V _max. In addition, acute exposure shifted steady state inactivation of V _max by 4–7 mV to more negative potentials. The decrease of V _max was frequency- and concentration-dependent. Halfmaximal inhibition (IC₅₀) of V _max by AM was affected by K⁺-induced membrane depolarization (in 4 mmol/1 K⁺,IC₅₀ 2.3 x 10^–4 mol/l; in 8 mmol/1 K⁺, IC₅₀ 9 x 10^–5 mol/1). Frequency-dependentinhibitionof closed Na⁺ channels by AM was demonstrated and AM increased the time constant for recovery from Na⁺ channel blockade. Depression of PF plateau by AM was similar to the effects of tetrodotoxin (TTX). Finally, AM depolarized RMP of PM exposed to low K⁺. The multiplicity of changes suggests that AM exerts inhibitory effects on a number of ionic current components, including at least fast Na⁺ current, slow inward current, TTX-sensitive plateau and outward K⁺ currents. Possible implications with respect to the broad spectrum of antiarrhythmic activity exhibited by AM are considered.Part of the study was carried out at the Department of Medicine (Division of Cardiology, Reingold ECG Center), Northwestern University Medical School, Chicago, 1L 60611, USA Send offprint requests to Masahiro Aomine at the above address     

3H-noradrenaline and 3H-5-hydroxytryptamine release from rat brain slices and its presynaptic alpha-adrenergic modulation after long-term desipramine pretreatment

Summary The electrically-evoked release of ³H-noradrenaline from superfused cortex and hippocampus slices was strongly enhanced (by about 50%) after long-term (4 weeks) pretreatment with desipramine (10 mg/kg, twice daily). The release-enhancing effect of the -receptor antagonist phentolamine was significantly reduced but the inhibitory effects of exogenous noradrenaline and clonidine on ³H-noradrenaline release were virtually unchanged after chronic desipramine treatment.The K⁺-induced release of ³H-noradrenaline from superfused synaptosomes obtained from rats pretreated with desipramine was about 25% higher than that from synaptomes of control animals. However, noradrenaline inhibited the K⁺-induced synaptosomal ³H-noradrenaline release to the same extent (viz. by about 55%) in both cases.The release of ³H-5-hydroxytryptamine induced by 26 mM K⁺ from cortex and hippocampus slices was not affected by chronic pretreatment with desipramine. In addition, no change was observed in the inhibitory effect of noradrenaline on ³H-5-hydroxytryptamine release.It is concluded that long-term pretreatment with desipramine leads to selective changes in the basic mechanism(s) of noradrenaline release rather than to changes in the sensitivity of presynaptic -adrenoceptors.     

Haloperidol affects coupling between QT and RR intervals in guinea pig isolated heart

Prolonged QT interval is an independent risk factor for development of ventricular arrhythmias. Haloperidol is one of the drugs inducing QT prolongation. Previous studies showed that haloperidol affects not only QT duration but also heart rate (RR interval). The present work focused on relationship between QT and RR and its changes under acute and chronic haloperidol administration. The study included 14 male guinea pigs divided into control and haloperidol-treated group. After 21-days administration of haloperidol or vehiculum, electrograms in isolated hearts were recorded. QT/RR and dQT/dRR coupling were calculated. Chronic haloperidol administration significantly decreases the coupling between QT and RR. Acute haloperidol exposure significantly decreases the dQT/dRR coupling in both treated and untreated guinea pig hearts. Flatter QT/RR relationship reveals a lack of QT adaptation to increased heart rate. It should be emphasized that in such situation ECG recording will not show significant QT prolongation evaluated according to clinical rules. However, if QT interval does not adapt to increased heart rate sufficiently, the risk of ventricular arrhythmias may be increased despite practically normal QT interval length. The results are supported by findings in biochemical analyses, which proved eligibility of the used model.     

Nociceptin inhibits noradrenaline release in the mouse brain cortex via presynaptic ORL1 receptors

A fourth type of opioid receptor, termed ORL₁, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [³H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca²⁺ 1.3 mM into Ca²⁺-free K⁺-rich (15 mM) medium. Nociceptin 1 μM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 μM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 μM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC₅₀ 7.5) was shifted to the right by the non-selective ORL₁ receptor antagonist naloxone benzoylhydrazone and the selective ORL₁ receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ (pA₂ 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ caused an inhibition by maximally 35% (pEC₅₀ 7.0; intrinsic activity α 0.45). The inhibitory effect of [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca ²⁺ -evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL₁ receptors, which are located presynaptically on noradrenergic neurones. Received: 19 June 1998 / Accepted: 17 July 1998     

A cumulative dose-response technique for the characterization of presynaptic receptors modulating [3H]noradrenaline release from rat brain slices

A cumulative dose-response technique was developed for the characterization of presynaptic receptors involved in the modulation of [³H]noradrenaline (NA) release from rat hippocampus slices, using continuous K⁺ (20 mM) depolarization. The results obtained with this technique were compared with those obtained using a repetitive K⁺ stimulation procedure. The release of [³H]NA induced by continuous K⁺ stimulation as well as that caused by repetitive K⁺ stimulation was strongly Ca²⁺-dependent and consisted for more than 90% of unmetabolized [³H]NA. Using continuous K⁺ stimulation it was demonstrated that the presynaptic inhibition of ³H-NA release by exogenous NA reached a maximum 10 min after addition of NA. The inhibitory effect of NA appeared to be independent of the time of addition, suggesting that the sensitivity of the presynaptic α-adrenoceptors remained unchanged during the experiment. Cumulative dose-response curves were recorded by the successive addition, at 10 min intervals, of increasing concentrations of NA. It was shown that continuous stimulation and repetitive K⁺ stimulation were basically similar with regard to the characteristics of the resulting [³H]NA release as well as its presynaptic α-adrenoceptor-mediated modulation by exogeneous NA. However, the cumulative dose-response technique, which can be carried out only using continuous K⁺ stimulation, makes it possible to determine more rapidly and also more accurately the apparent affinities and intrinsic activities of drugs towards receptors involved in the modulation of neurotransmitter release from brain slices.     

Facilitation of noradrenaline release by gallamine in the rat salivary gland

Summary The effect of gallamine on spontaneous and stimulation-evoked overflow of tritium was studied in the submandibular gland of the rat. The gland was perfused retrogradely and labeled with³H-noradrenaline. The stimulation-evoked (1 Hz for 60 s) overflow of tritium was facilitated by increasing concentrations of gallamine (0.3–20 mM). None of the concentrations of gallamine increased the spontaneous overflow of the tritium. The facilitatory effect of gallamine was observed in 0.3 to 5 mM calcium medium; the maximum facilitation was observed at the normal concentration of calcium (2.5 mM). The facilitatory effect of gallamine was inversely related to the frequency of stimulation (10-fold facilitation at 1 Hz and 3-fold at 10 Hz).Stimulation of the salivary gland by a single pulse (1 ms duration) in the normal medium did not evoke an overflow of tritium; however, the same stimulus produced a marked increase in the overflow in the presence of gallamine.The facilitatory action of gallamine on the release of sympathetic transmitter is ascribed to the enhanced availability of calcium ions to the secretory process resulting from blockade of potassium conductance during nerve activity.     

Cyclic AMP and alpha-receptor-mediated modulation of noradrenaling release from rat brain slices.

Oxymetazoline (an alpha-receptor agonist) reduced and phentolamine (an alpha-receptor antagonist) increased depolarization-induced release (potassium or electrical field stimulation) of 3H-noradrenaline (NA) from superfused neocortical slices in a dose-dependent way. Inhibition of phosphodiesterase also reduced NA release; this effect could be reversed by phentolamine. Phosphodiesterase inhibition potentiated the effect of oxymetazoline. It is suggested that stimulation of presynaptic alpha-receptors may reduce NA release up to about 60% and that increased cyclic AMP formation might be involved in this modulation.     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

Modulation of noradrenaline release from the sympathetic nerves of human right atrial appendages by presynaptic EP3- and DP-receptors

Strips of human right atrial appendages were preincubated with [³H]noradrenaline and then superfused with physiological salt solution containing inhibitors of uptake₁ and uptake₂. Tritium overflow was evoked by transmural electrical stimulation (standard frequency: 2 Hz). Prostaglandin E₂ (PGE₂) inhibited the electrically evoked tritium overflow; at the highest concentration investigated, tritium overflow was reduced by about 80% and the pIC_50% value was 7.14. The effect of PGE₂ was not affected by rauwolscine, which, by itself, increased the evoked overflow. Naproxen failed to affect the evoked tritium overflow and its inhibition by PGE₂. The inhibitory effect of PGE₂ on the electrically evoked tritium overflow was mimicked by prostaglandin E₁, the EP₁/EP₃-receptor agonist sulprostone and the EP₂/EP₃-receptor agonist misoprostol with the rank order of potency (pEC_50%): sulprostone (7.68) > misoprostol (7.10) > PGE₁ (6.39). In contrast, PGF_2α, the IP/EP₁-receptor agonist iloprost and the stable thromboxane A₂ analogue U46619 (9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F_2α) did not change evoked tritium overflow. PGD₂ caused facilitation. These results suggest that the sympathetic nerve fibres innervating human atrial appendages are endowed with presynaptic inhibitory EP₃ and facilitatory DP-receptors. The EP₃-receptors appear not to be tonically activated and do not interact with the α₂-autoreceptors. Received: 11 May 1998 / Accepted: 29 July 1998