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     

Modulation of calcium currents via α2-adrenoceptors in embryonic chick sympathetic neurons

Summary In order to gain insight into the mechanism of the autoinhibition of noradrenaline release, the present study explores the effects of substances acting at various adrenoceptor-subtypes on voltage-activated Ca ²⁺ currents. Experiments were carried out on cultured embryonic chick sympathetic neurons using the patch clamp technique.Ca²⁺ currents associated with a (fully activating) depolarizing 150 ms voltage step to 0 mV were reduced by noradrenaline and the two a2-adrenoceptor agonists UK 14,304 and clonidine, predominantly during the early phase of activation. We quantified these effects by measuring Ca²⁺ current amplitudes in the absence and presence of substances 10 ms after the beginning of the depolarization. Noradrenaline effects were maximal at 5 µmol/l, causing a 28% depression of the current. Half-maximal effects (IC₅₀) were apparent at 0.7 µmol/l. UK 14,304 was equipotent to noradrenaline (IC₅₀: 0.5 µmol/l; maximal effect: 26% depression). Clonidine, while active in the same range of concentration (IC₅₀: 0.6 µmol/l), had a smaller maximal effect (20% depression). Methoxamine and isoprenaline, on the other hand, did not significantly reduce the Ca ²⁺ current at 10 µmol/l. The noradrenaline-induced inhibition was attenuated by yohimbine (1 µmol/I). Neither prazosin (1 µmol/l) nor propranolol (1 µmol/l) interfered with the effect of noradrenaline.These results indicate a reduction of Ca ²⁺ influx via ₂-adrenoceptors and suggest that the autoreceptor-mediated inhibition of transmitter release in embryonic chick sympathetic neurons operates through the modulation of Ca²⁺ channels. Send offprint requests to S. Boehm at the above address     

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.     

Role of cyclic AMP in the release of noradrenaline from isolated rat atria

Summary The possible role of cyclic AMP (cAMP) on tritium overflow evoked by stimulation of the cardioaccelerant nerves was studied in rat atria preincubated with [³H]-noradrenaline. Addition of the activator of adenylate cyclase forskolin (1 µmol/l), or of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMx, 100 µmol/l), did not affect both basal and evoked overflow. However, in the presence of the ₂-adrenoceptor antagonist yohimbine (0.03 µmol/l) both forskolin and IBMx increased the stimulation-induced transmitter overflow by 49% and 141%, respectively (compared to yohimbine 0.03 µmol/l). Thus, in rat atria the cAMP-dependent facilitation of noradrenaline release is only present when the autoinhibition exerted by activation of prejunctional ₂-adrenoceptors is blocked. Propranolol (0.1 µmol/l) that did not produce any effect on noradrenaline release markedly reduced the facilitatory response induced by forskolin in the presence of yohimbine. When rats were pretreated with the ₂-adrenoceptor agonist clenbuterol (0.3 mg · kg^–1, s. c., twice daily, 14 days), a treatment which desensitizes -adrenoceptor-me-diated facilitation of noradrenaline release (Kazanietz and Enero 1989), the facilitatory effect of forskolin and IBMx in the presence of yohimbine was abolished. The results indicate that in rat atria the effect of forskolin and IBMx on noradrenaline release are only to be observed after blockade of presynaptic ₂-adrenoceptor autoinhibition. -adrenoceptor blockade or clenbuterol pre-treatment decreases the facilitatory response to forskolin and hence prejunctional -adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase. Correspondence to M. A. Enero at the above address     

Effects of low temperature and pharmacological interventions on the responses of the isolated guinea-pig trachea

Summary Cooling the guinea-pig isolated trachea from 37°C to 20°C virtually abolished the response to CaCl₂ (in K⁺-depolarized tissues) and depressed that to histamine (about 75% reduction), KCl and 5-hydroxytryptamine (around 50% inhibition) while the response to acetylcholine remained unaffected. A further cooling to 10°C was necessary to inhibit acetylcholine-induced contractions. Hyporesponsiveness to spasmogens by cooling was not associated with subsensitivity (increased EC₅₀) except for 5-hydroxytryptamine. Contractile responses to KCl (50 mmol/l), histamine (1 mmol/l) and 5-hydroxytryptamine (0.1 mmol/l) in a Ca²⁺-free EGTA (0.1 mmol/l)-containing solution were inhibited by cooling to 20°C but responses to acetylcholine (1 mmol/l) in the same experimental conditions were not affected. Cooling to 20°C after treatment with an antagonist (ouabain 10 µmol/l, amiloride 0.1 mmol/l or vanadate 0.1 mmol/l) or after incubation in K⁺-free medium or low Na⁺ (25 mmol/l) solution produced the same or greater inhibitions of tracheal responses to spasmogens than cooling alone. The guinea-pig trachea treated with phorbol 12,13-diacetate (PDA; 1 µmol/l) and cooled to 20°C responded to spasmogens similarly to a trachea untreated with PDA at 37°C. In contrast, PDA (1 µmol/l) did not counteract the depressed responsiveness to histamine of ouabain (10 µmol/l)- or amiloride (0.1 mmol/l)- treated tracheal strips at 37°C. PDA (1 mol/l) enhanced tracheal contractions caused by KCl (50 mmol/l) in Ca²⁺-free medium at 20°C but failed to augment those to histamine in the same conditions. PDA (0.1 or 1 µmol/l) did not change the concentration-response curve for Ca²⁺ in skinned trachea. In conclusion, low temperature inhibits the responses to spasmogens in the guinea-pig trachea. Probably, reduced temperature interferes with extracellular and intracellular sources of Ca²⁺ which are differently affected by various spasmogens. Alteration by cooling of the electrogenic Na⁺ pump, Na⁺/Ca²⁺ exchange system, and Ca²⁺-ATPase may participate in the decrease of tracheal responsiveness to agonists. Reversion by PDA of the inhibitory effect of low temperature suggests a role for protein kinase C in the cooling-induced changes of tracheal responses. Correspondence to J. Cortijo 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-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     

Studies on alpha adrenoceptors in guinea-pig peripheral lung strips

Summary Contractile responses of guinea-pig peripheral lung strips to noradrenaline were determined in the presence of propranolol (2.5 × 10^–6 mol/l). All strips (n = 44) contracted to noradrenaline and a geometric mean EC₅₀ of 1.4 × 10^–6 mol/l (1.1 × 10^–6 mol/l, 1.8 x 10^–6 mol/l 95% confidence limits) was obtained. Contractions were antagonised by phentolamine (5 × 10^–7–10^–5 mol/l) and by prazosin (10–10^–7 mol/l). Dose-ratios (DR) were calculated and log (DR-1) was plotted against log concentration of antagonist to yield slopes (± SE) of 0.84 ± 0.14 and 0.73 ± 0.22 respectively which were not significantly different from unity. A pA₂ value (± SE) of 6.7 ± 0.2 was obtained for phentolamine and 7.5 ± 0.1 for prazosin. Yohimbine (10^–7–10^–5 mol/l) did not significantly affect the maximal tension generated or the EC₅₀ values for noradrenaline. These results suggest that ₁ adrenoceptors are mediating the contractile responses to noradrenaline. In the presence of cocaine (10^–5 mol/l, n=18), normetanephrine (2 × 10^–5 mol/l, n = 15), hydrocortisone (2.5 × 10^–5 mol/l, n = 15) and normetanephrine (2 × 10^–5-5 mol/l) plus cocaine (10^–6 5 mol/l, n=15) pA₂ values for phentolamine of 6.9, 6.7, 6.6, and 6.3 respectively were obtained. Since these pA₂ values are not significantly different from 6.7, it is unlikely that this original pA₂ value, which is lower than values obtained with phentolamine at -adrenoceptors in other tissues, may be explained by neuronal or extraneuronal uptake of noradrenaline. A possible explanation may be that more than one population of -adrenoceptors contribute to changes in tension in peripheral lung strips. Send offprint requests to J. P. Seale 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     

Some degree of overlap exists between the K+-channels opened by cromakalim and those opened by minoxidil sulphate in rat isolated aorta

Summary The effects of the K⁺ channel opening drugs minoxidil sulphate and cromakalim, on ⁴²K⁺ and ⁸⁶Rb⁺ efflux and on vasorelaxation in rat isolated aorta, were compared. In rat aortic rings precontracted with noradrenaline (100 nmol/l), minoxidil sulphate and cromakalim concentration-dependently inhibited induced tension by up to 90%, with pD₂ values of 7.35±0.1 and 7.17±0.1, respectively. Glibenclamide (300 nmol/l), produced 2200- and 19-fold rightward shifts in the concentration-relaxation curves to minoxidil sulphate and cromakalim, respectively, without an effect on the maximum relaxation.Both minoxidil sulphate and cromakalim increased the efflux of ⁴²K⁺ and ⁸⁶Rb⁺ from aorta in a concentration-dependent manner, with midpoints in the µmol/l range; the maximum efflux induced by minoxidil sulphate being approximately one tenth of that induced by cromakalim. The ratio of stimulated ⁸⁶Rb⁺/⁴²K⁺ efflux increased from 0.22 to 0.48 with increasing cromakalim concentrations, but was approximately constant (0.39) when the minoxidil sulphate concentration was varied. In the presence of minoxidil sulphate, the effects of cromakalim on ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited in a concentration-dependent manner, by up to 60%. In the continuing presence of cromakalim (300 nmol/l), minoxidil sulphate (10 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 µmol/l) inhibited the ⁸⁶Rb⁺ efflux stimulated by additional superfusion of cromakalim (1 µmol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 µmol/l)- and cromakalim (1 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux in a concentration-dependent manner with IC₅₀ values of approximately 80 nmol/l.In conclusion, the efflux data suggest that considerable overlap exists between the channels opened by minoxidil sulphate and those opened by cromakalim in rat aorta. Minoxidil sulphate has a weak efficacy as a K⁺ channel opener, and may act to open a homogeneous population of K⁺ channels. In contrast, the actions of cromakalim (1 µmol/l) are associated with large increases in tracer efflux, which are probably mediated via a heterogeneous population of K⁺ channels. However, only a small proprtion of this induced efflux appears to be required for relaxation. The differential inhibition by glibenclamide of the vasorelaxant effects of minoxidil sulphate and cromakalim may result from (a) the partial agonist properties of minoxidil sulphate in opening K⁺ channels and/or (b) additional mechanisms of vasorelaxation, which differ in their sensitivity to glibenclamide. Send offprint requests to U. Quasi 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     

Extraneuronal uptake of noradrenaline in rabbit dental pulp: evidence of identity with uptake

Summary The extraneuronal removal and disposition of noradrenaline in rabbit dental pulp was examined in view of earlier evidence that the tissue possessed an extra-neuronal uptake process resembling neuronal uptake₁. Pulp, which had been depleted of sympathetic nerves by homolateral superior cervical ganglionectomy, was incubated in vitro with ³H-noradrenaline in low concentrations (0.025 or 0.18 mol/l). When the metabolising enzymes (monoamine oxidase, catechol-O-methyl transferase) were active, ³H retention by the denervated pulp, as indicated by the ³H content after the tissue had been washed for 30 min following incubation with ³H-noradrenaline, was less than 30% of that of the innervated pulp. When the enzymes were inhibited, retention rose to approximately 30% of that of the innervated pulp. Analysis of the time course of the ³H efflux indicated that the ³H-noradrenaline in the denervated pulp had accumulated in a single compartment characterised by a t_1/2 for efflux of several hours. Accumulation did not occur under Na⁺-free conditions, and was inhibited by desipramine (IC₅₀ < 0.03 mol/l) and by substrates of neuronal uptake₁. Mean IC₅₀, values of the latter were very similar to those for inhibition of neuronal uptake₁ and comprised (in mol/l): (+)amphetamine (0.29), dopamine (0.31), tyramine (0.39), (–)noradrenaline (0.70), (–)adrenaline (1.50), 5-hydroxytryptamine (20) and bretylium (35). Uptake₂ inhibitors were less active (O-methyl isoprenaline, IC₅₀ = 60 mol/l) than uptake₁ inhibitors, or were without inhibitory effects at the concentrations tested (hydrocortisone, 210 mol/l; 2-methoxy oestrone, 10 mol/l).The effects of Na⁺ omission, of (+)amphetamine, and of O-methylisoprenaline on ³H-normetanephrine formation (measured in the absence of catechol-O-methyl transferase inhibition) matched their effects on ³H-noradrenaline accumulation. The results provide strong support for the presence in rabbit dental pulp of extraneuronal uptake₁ which is linked with catechol-O-methyl transferase in the removal of noradrenaline. Send offprint requests to D. A. S. Parker 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     

Blockade of α2-adrenoceptors increases opioid μ-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones

Summary In pontine slices of the rat brain, the frequency of spontaneous action potentials of locus coeruleus (LC) neurones was recorded extracellularly. Noradrenaline 0.1–100 mol/l, UK 14,304 0.01–100 nmol/l, [Met⁵]-enkephalin 1–10,000 nmol/l and [D-Ala², D-Leu⁵]enkephalin 0.1–1,000 nmol/l, all depressed the firing rate. Rauwolscine 1 mol/l antagonized the effects of both noradrenaline and UK 14,304, but potentiated the effects of [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin. Idazoxan 1 mol/l acted in a similar manner. Prazosin 1 mol/l did not change the effects of either noradrenaline or [Met⁵]enkephalin. Naloxone 0.1 mol/l antagonized both [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin, but failed to alter the effects of either noradrenaline or UK 14,304. Rauwolscine, idazoxan and prazosin, all 1 mol/l, as well as naloxone 0.1 mol/l, did not influence the firing rate when given alone. Desipramine 1 mol/l inhibited the discharge of action potentials in a rauwolscine-antagonizable manner. Noradrenaline 10 mol/l produced the same depression of firing, both in the presence of noradrenaline 1 mol/l and [Met⁵]enkephalin 0.03 mol/l. Likewise, the effect of [Met⁵]enkephalin 0.3 mol/l was the same, irrespective of whether it was added to a medium containing [Met⁵]enkephalin 0.03 mol/l or noradrenaline 1 mol/l. The spontaneous activity of LC neurones is inhibited by somatic ₂-adrenoceptors and opioid -receptors. We suggest that the two receptors interact with each other at a site located between themselves and not in the subsequent common signal transduction system.Send offprint requests to: P. Illes at the above address     

The effects of quinpirole and fenoldopam on the potassium-evoked overflow of endogenous dopamine and noradrenaline in dog mesenteric arteries

Summary (1) Dopamine and noradrenaline overflow from the main trunk of the dog mesenteric artery and its proximal branches, elicited by K⁺ (52 mmol/l), was measured by high pressure liquid chromatography with electrochemical detection. (2) Quinpirole (0.1, 1 and 10 nmol/l) produced a concentration dependent reduction of dopamine and noradrenaline overflow in both segments of the mesenteric artery. The inhibitory effect of quinpirole (10 nmol/l) on amine overflow was antagonized by sulpiride (1 mol/l) but not by phentolamine (0.2 mol/l) or the selective dopamine (DA₁), antagonist SK&F 83566 (1 ol/l). (3) Fenoldopam (0.1 and 1 mol/l) did not alter dopamine and noradrenaline overflow from both segments of the mesenteric artery; only 10 mol/l fenoldopam was found to increase the overflow of dopamine and noradrenaline in both segments of the mesenteric artery. This effect of fenoldopam on amine overflow was not altered by the addition to the perifusion fluid of SK&F 83566 (1 ol/l). (4) Clonidine (100 nmol/l) significantly reduced amine overflow from both segments of the mesenteric artery and this effect was antagonized by fenoldopam (10 mol/l) (5) These results suggest that quinpirole inhibits sympathetic neurotransmission through the activation of prejunctional dopamine receptors of the DA₂ subtype. The facilitatory effect of fenoldopam (10 mol/l) on amine release appears to be mediated through the blockade of prejunctional ₂-adrenoceptors. Send offprint requests to P. Soares-da-Silva at the above address     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Evidence for uptake1-mediated efflux of catecholamines from pulmonary endothelial cells of perfused lungs of rats

Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake₁. In noradrenergic neurones, it has been shown that uptake₁ is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake₁, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells.The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake₁ on the efflux of ³H-noradrenaline and ³H-dopamine from rat lungs were measured. Isolated; perfused lungs of rats (monoamine oxidase and catechol-0-methyltransferase inhibited) were loaded with ³H-(–)noradrenaline or ³H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free: Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake₁ for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min^–1 and 0.0466 min^–1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LICl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake₁ substrates, dopamine (1 and 3 mol/l) and adrenaline (40 mol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mol/l) had a similar effect on the efflux of dopamine. The ^uptake 1 inhibitors, imipramine (3 and 10 mol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs.These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake₁ transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake₁. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake₁.Abbreviations COMT Catechol-O-methyltransferase - FRL Fractional rate of loss - K _m Michaelis or half-saturation constant - t _out rate constant for efflux - k _uptake rate constant for uptake - MAO monoamine oxidase - t _/12 half-time for efflux - U-0521 3,4-dihydroxy-2-methylpropiophenone - V _max maximal rate of uptake Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Scarcella et al. 1993).     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.

     

Altered serum protein binding of carbamazepine in disease states associated with an increasedα 1-acid glycoprotein concentration

Summary The protein binding of carbamazepine (CBZ) in vitro was assessed in sera from 47 patients with various diseases known to alter ₁-acid glycoprotein (AAG) concentration and from 20 drug-free normal control subjects. In the patient group, AAG and albumin (HSA) concentrations ranged from 6 to 74 µmol/l and from 377 to 652 µmol/l, respectively; in the controls, protein concentrations were less variable, ranging from 11 to 26 µmol/l for AAG and from 623 to 754 µmol/l for HSA. In both the patient and the combined patient and control groups, free CBZ fractions were inversely correlated with the serum AAG concentration (r=–0.62). No significant relationship could be found between the free CBZ fraction and the serum HSA concentration. The free CBZ fraction was moderately but significantly decreased in patients with AAG levels above 26 µmol/l (the highest value found in controls) as compared either to patients with a normal AAG concentration or to control subjects (19±5% vs 23±4% and 23±2%), despite the finding of a higher HSA concentration in the control group. The data confirm AAG as an important determinant of interindividual variability in serum CBZ binding.     

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney

Summary The present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [³H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC₃₀=4.5×10^–8 mol/l), both 5-carboxamidotryptamine (IC₃₀=8×10^–9 mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC₃₀=2.5×10^–7 mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC₅₀=4×10^–9 mol/l), metergoline (IC₅₀=4×10^–8 mol/l) and methysergide (IC₅₀=1.3×10^–7 mol/l) but not by cyproheptadine, ketanserin, mesulergine, (–)-propranolol, (±)-pindolol, (±)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT₁-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT_1A, 5-HT_1B or 5-HT_1C subtypes.Preliminary accounts of this work were presented to the British Pharmacological Society in London (December, 1984) and Southampton (July, 1985)