Effects of hypoxia on contractile responses of rabbit aortic strips to transmural electrical stimulation

Summary To clarify the effects of hypoxia on adrenergic transmission, we examined the contractile responses of isolated rabbit aortic strips to electrical stimulation, the concentration-response relationships for noradrenaline and KCl, and the electrical stimulation-evoked overflows of total [³H] and [³H]noradrenaline from strips preloaded with [³H]noradrenaline in media equilibrated with gas mixtures containing various concentrations of 0₂. Contractile responses to electrical stimulation were completely inhibited by tetrodotoxin and -adrenoceptor antagonists such as phentolamine and phenoxybenzamine, but were not affected by indomethacin. When the concentration of O₂ in the gas mixture was decreased from 95% to 20%, the contractile responses to electrical stimulation remained unchanged, but as the concentration of OZ was further decreased, the responses were inhibited concentration-dependently. At 0% O₂, the response was inhibited by about 80% when compared with control values obtained at 95% O₂, and the electrical stimulation-evoked overflows of total [³H] and [³H]noradrenaline into the superfusates were decreased by about 55%. At 0% 02, the concentration-response curve for exogenous noradrenaline was shifted to the right about 50-fold and the maximum response was decreased by 25%. The maximum contractile responses of aortic strips from animals pretreated with reserpine or 6-hydroxydopamine to high KCl were decreased slightly (about 15%). These results suggest that inhibition of adrenergic transmission under hypoxic conditions is mainly the result of a decrease in the stimulus-evoked release of noradrenaline and of a decrease in the affinity of -adrenoceptor for noradrenaline and/ or inhibition of signal transduction mechanisms, although hypoxia also causes a slight decrease in the contractility of vascular smooth muscle.This work was supported by a Grant-in-Aid for New Drug Development from the Ministry of Health and Welfare of Japan and by a Grant from Smoking Research Foundation, Japan Send of fprint requests to S. Miwa at the above address     

Pharmacological characterization of the inhibitory effects of neurotensin on the rabbit ileum myenteric plexus preparation.

1 [³H]-amezinium is taken up selectively into noradrenergic axons and their transmitter-storing vesicles and is released from these axons by action potentials. We used it as a non-α-adrenergic marker in order to study the α-adrenergic autoinhibition of noradrenaline release.

2 Rat occipitocortical slices were preincubated with [³H]-amezinium 0.03 μM and then superfused and stimulated electrically (3 Hz for 3 min). The stimulation-evoked overflow of tritium was measured in six groups of slices: from saline-pretreated rats; from saline-pretreated rats, the slices being exposed to exogenous noradrenaline before preincubation with [³H]-amezinium; from saline-treated rats, slices from which were exposed simultaneously to noradrenaline and cocaine before preincubation with [³H]-amezinium; from rats in which noradrenaline stores had been depleted by pretreatment with α-methyltyrosine (α-MT); from α-MT-treated rats, the slices being exposed to noradrenaline before preincubation with [³H]-amezinium; and from α-MT-treated rats, slices from which were exposed to noradrenaline plus cocaine before preincubation with [³H]-amezinium.

3 The stimulation-evoked overflow of tritium, expressed as a percentage of the tritium content of the tissue, was 1.15% in slices from saline-pretreated rats, and was similar in slices from saline-pretreated rats after exposure to noradrenaline or noradrenaline plus cocaine. It was 2.56% in slices from α-MT-treated rats, 1.20% from α-MT-treated rats after exposure to noradrenaline, and 2.88% from α-MT-treated rats after exposure to noradrenaline plus cocaine.

4 Yohimbine 0.1 and 1 μM increased the stimulation-evoked overflow of tritium in slices from all groups of saline-pretreated rats and in those slices from α-MT rats that had been in contact with exogenous noradrenaline. Yohimbine did not change the evoked overflow in slices from α-MT rats that had not been exposed to noradrenaline, or had been exposed to noradrenaline plus cocaine.

5 Clonidine 0.01-1 μM decreased the stimulation-evoked overflow of tritium moderately in slices from saline-pretreated rats, markedly in slices from α-MT-treated rats, and moderately again when the latter slices had been exposed to noradrenaline.

6 It is concluded that the action potential-evoked release of [³H]-amezinium as well as the modulation of this release by yohimbine and clonidine depend on the presence or absence of α-adrenergic autoinhibition caused by the co-secretion of noradrenaline. When there is co-secretion of noradrenaline, the evoked release of [³H]-amezinium is relatively small, yohimbine increases the release, and clonidine can cause only moderate inhibition. When there is no or very little co-secretion of noradrenaline, the evoked release of [³H]-amezinium is at least doubled, yohimbine causes no further increase and clonidine produces strong inhibition.

     

Leucine-enkephalin- and neuropeptide Y-modulation of [3H]noradrenaline release in the oviduct of mature and juvenile rabbits

1. The effects of leucine-enkephalin and neuropeptide Y (NPY) on [3H]noradrenaline release induced by electrical field stimulation were studied in the isthmic part of the oviduct of juvenile and mature rabbits. 2. [3H]noradrenaline and total tritium overflow in the presence of cocaine, corticosterone and hyoscine were determined by liquid scintillation spectrometry. 3. Tritium overflow evoked by electrical stimulation (1 or 4 Hz, 1 msec) was calcium dependent. [3H]noradrenaline content (measured by ion exchange chromatography) accounted for 85% of the total tritium overflow. 4. Leucine-enkephalin (1 microM) in the presence of the peptidase inhibitor bacitracin reduced the stimulation-evoked tritium overflow in mature rabbits by 26.1 +/- 1.6% and in juvenile rabbits by 11.9 +/- 1.9%. Naloxone (1 microM) antagonized the effect of leucine-enkephalin. 5. NPY (0.2 microM) reduced the evoked tritium overflow in mature rabbits by 23.4 +/- 2.4% and in juvenile rabbits by 17.2 +/- 4.3%. 6. It is concluded that leucine-enkephalin and NPY inhibited [3H]noradrenaline release in rabbit oviduct and the modulatory effect of leucine-enkephalin depends on maturity while NPY modulation is a more independent system.     

THE EFFECTS OF COCAINE AND LIGNOCAINE ON THE RAT RIGHT VENTRICLE IN VITRO

• 1 The effects of cocaine and lignocaine on the contractile responses to field stimulation and to exogenously applied agents, in the absence of other stimuli, have been investigated in the rat right ventricle using methods we have recently described (Doggrell & Vincent, 1981a). In addition the effects of ³H accumulation from (?)-[³H]-noradrenaline and on the spontaneous and field stimulation-induced overflow of ³H, following preloading of the tissue with (?)-[³H]-noradrenaline, are reported.
• 2 Cocaine, but not lignocaine, inhibited the accumulation of ³H from (?)-[³H]-noradrenaline. The spontaneous overflow of ³H, following preloading of the tissue with (?)-[³H]-noradrenaline was not altered by cocaine, 10μ, or lignocaine, 100μM. 10μM Lignocaine had no effect on the overflow of ³H evoked by field stimulation at 5Hz. Lignocaine, 100μM, increased and cocaine, 1 and 10μM, reduced the decline in evoked release of ³H. This effect of lignocaine probably represents a decrease in nerve excitability and that of cocaine inhibition of neuronal uptake of noradrenaline.
• 3 Cocaine, μM, reduced the rate of beat response to tyramine, 1μM, alone, probably by inhibiting the neuronal uptake process.
• 4 Cocaine, 1 and 10μM, had no effect on the contractile responses to field stimulation (at 2 and/or 5Hz). The rate of beat to (?)-noradrenaline or (?)-isoprenaline, 1μM, alone was decreased by cocaine, 10μM. Lignocaine, 10μM, reduced the force of contractions to field stimulation at 5 Hz and the responses to (?)-noradrenaline or (?)-isoprenaline alone. It is suggested that the inhibitory effects of cocaine and lignocaine on responses to (?)-isoprenaline in the rat right ventricle are due to a decreased postjunctional membrane excitability. The inability of 10μM cocaine to potentiate contractile responses to endogenous or exogenous (?)-noradrenaline as a consequence of the inhibition of neuronal uptake is also probably due in part to decreased post-junctional excitability of the right ventricle.

     

The effects of labetalol (AH 5158) on metabolism of 3H(-)-noradrenaline released from the cat spleen by nerve stimulation

The competitive α and β adrenoceptor antagonist labetalol, in concentrations up to 10^?4 M, produced a dose dependent increase in overflow of ³H and [³H]noradrenaline in the isolated blood perfused cat spleen following stimulation of the splenic nerves at a frequency of 10 Hz. Labetalol had no effect on the pattern of overflow of label following stimulation. In experiments in which the metabolism of [³H]noradrenaline released on nerve stimulation was examined, labetalol produced a concentration dependent increase in the percentage of [³H]noradrenaline and a decrease in the percentage of [³H]DOPEG in the venous blood following nerve stimulation. Production of [³H]COMT metabolites and [³H]DOMA was not affected. It is suggested that in the isolated blood perfused cat spleen labetalol produces the elevation of overflow and effects on noradrenaline metabolism by inhibition of neuronal uptake of noradrenaline. The drug has no detectable effects on the enzymes MAO or COMT or on extraneuronal uptake.     

The inhibitory action of pgf2α on release of [3H]noradrenaline enhanced by α2-adrenoceptor blockade,sodium-pump inhibition and 4-aminopyridine in the main pulmonary artery of the rabbit

Large concentrations of prostaglandin PGF_2α inhibited the stimulation (2 Hz) evoked release of [³H]noradrenaline from the isolated main pulmonary artery of the rabbit (the inhibition caused by 3 × 10^?5 M PGF_2α was 62%). Furthermore, PGF_2α inhibited the release evoked by stimulation when it was enhanced by different procedures. During blockade of presynaptic α₂-adrenoceptors by 3 × 10^?7 M yohimbine, which by itself enhanced the overflow of [³H]NA in response to stimulation, the inhibitory action of PGF_2α was more pronounced (78.2%). In tissue in which the Na⁺-pump was inhibited (K⁺-free treatment) where the overflow of ³H was markedly increased, PGF_2α exerted nearly equal inhibition of transmitter release to that observed in control experiments (64.3%). The inhibitory effect of PGF_2α on the stimulation-evoked release of [³H]NA was less pronounced (32.1%) in the presence of 10^?4M 4-aminopyridine (a blocker of K⁺ -channels).     

Effects of S9977 on adrenergic neurotransmission

1. Experiments were designed to determine whether or not the putative promnesic drug S9977 (1,3,7-trimethyl 8-[3-(4-diethylaminocarbonyl-1-piperazinyl) 1-propyl]-3,7-dihydro (1H)2,6-purinedione hydrochloride) affects peripheral adrenergic neurotransmission.2. Rings of canine saphenous veins (without endothelium) were suspended for isometric tension recording in conventional organ chambers filled with modified Krebs-Ringer bicarbonate solution. The adrenergic nerve endings were activated with electrical impulses (9 V, 2 msec, 0.25–8 Hz).3. At 10⁻⁵ M, S9977 significantly reduced the contraction to 0.25, 0.5 and 1 Hz. The compound did not affect the response to higher stimulation frequencies or to exogenous noradrenaline. The inhibitory effect of S9977 was prevented by methiothepin, and not affected by atropine or 8-phenyltheophylline.4. Helical strips of canine saphenous veins were incubated with [³H]noradrenaline and suspended for superfusion and isometric tension recording. Under basal conditions, S9977 (10⁻⁴ M) augmented, the total ³H-overflow which was due mainly to an augmented overflow of [³H]deoxyphenylglycol (DOPEG); the extraneuronal metabolites 3,4-dihydromandelic acid (DOMA) and 3-methoxy-4-hydroxymandelic acid (VMA) were reduced.5. During electrical stimulation of the adrenergic nerves, S9977 (10⁻⁴ M) augmented the total ³H-overflow but reduced the contractile response; the evoked overflow of [³H]noradrenaline was not significantly affected.6. These experiments suggest that S9977 the displacement of noradrenaline from the adrenergic varicosities; most of the displaced transmitter is metabolized by intraneuronal monoamine oxidase before reaching the junctional cleft. In addition, S9977 exerts an inhibitory effect on the extraneuronal metabolism of catecholamines. S9977 does not inhibit the exocytotic release of the adrenergic neurotransmitter.     

Effect of cocaine on tritium overflow evoked from vasa deferentia previously loaded with [3H]noradrenaline by stimulation using different types of electrode.

In mouse and guinea-pig vasa deferentia previously incubated with [3H]noradrenaline, electrical stimulation applied through parallel electrodes (transmurally) increased overflow of tritium 2- to 5-fold above the resting value. Electrical stimulation applied using methods involving more substantial conduction of nerve impulses in neuronal elements in the tissues evoked a tritium overflow which was smaller (70%) than that evoked by transmural stimulation. Cinchocaine (25 microM), tetrodotoxin (0.5 microM) or the absence of calcium effectively abolished evoked overflow in both tissues whichever method of stimulation was used. In mouse vas deferens, cocaine (10 microM) did not alter overflow evoked by either transmural or axonal stimulation while 100 microM produced a reduction. In guinea-pig vas deferens, cocaine (10 microM) produced a statistically significant increase in evoked overflow of about 50% or more with both transmural and axonal stimulation. As in mouse vas deferens, 100 microM cocaine produced a reduction. It is concluded that the action of cocaine is independent of these methods of stimulation and that some difference in the arrangement of the noradrenergic nerves in the two species may account for the differential effect of cocaine observed.     

Cannabinoid receptor-mediated inhibition of dopamine release in the retina

The possible occurrence of cannabinoid (CB) receptors was studied on superfused guinea-pig retinal discs preincubated with [3H]dopamine or [³H]noradrenaline. Tritium overflow was evoked either electrically (3 Hz) or by re-introduction of Ca²⁺, 1.3 mM after superfusion with Ca²⁺-free medium containing K⁺ 30 rnM. The accumulation of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) was inhibited by the selective inhibitor of the neuronal dopamine transporter GBR-12909 (pIC_50% 7.29 and 7.41, respectively) but not by the selective inhibitor of the neuronal noradrenaline transporter desipramine (1 M). The electrically or Ca²⁺-evoked tritium overflow in retinal discs preincubated with [³H]DA or [³H]NA was reduced by the CB receptor agonists CP-55,940 and WIN 55,212-2 (pIC_50% in discs preincubated with [³H]NA, electrical stimulation: 7.03 and 6.70, respectively) but not affected by the inactive S(–)enantiomer of the latter, WIN 55,212-3 (up to 10 M). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist SR 141716 (apparent pA₂: 8.29) which, by itself, increased the evoked overflow. The facilitatory effect of SR 141716 was not affected by GBR-12909 and the dopamine receptor antagonist haloperidol. In conclusion, the dopaminergic neurones of the guinea-pig retina can be labelled by both [³H]DA and [³H]NA. Transmitter release from the dopaminergic neurones is inhibited by activation of cannabinoid receptors of the CB₁ type, which appear to be tonically activated by an endogenous CB receptor ligand.     

Opioid receptor sub-types involved in the control of transmitter release in cortex of the brain of the rat

Electrical stimulation (3 Hz, 2msec duration, 5–12 V for 2 min every 20min) of cortical slices from the rat, previously incubated with [³H]noradrenaline, evoked a release of tritium which was inhibited by morphine, normorphine, Tyr-d-Ala-Gly-MePhe-NH(CH₂)₂OH (RX783006) and d-Ala²-d-Leu⁵-enkephalin (pIC₃₀ 5.90, 6.32. 7.45 and 6.74 respectively). Naloxone did not affect the release of tritium when given alone but antagonised the actions of the opioids, giving a K_e value of about 3 nM irrespective of the particular agonist used, which suggests an action at mu receptors. The delta opioid receptor blocker, ICI154129, antagonised the opioids only in large concentrations (K_e 21300nM). In slices previously incubated with [³H]5-hydroxytryptamine, electrical stimulation increased overflow of tritium but neither naloxone nor the opioid agonists affected evoked overflow of tritium at concentrations which were effective in slices incubated with [³H]noradrenaline. It is concluded that stimulation of mu opioid receptors may inhibit release of noradrenaline from central noradrenergic neurones and that these receptors are not present in significant numbers on neurones releasing 5-hydroxytryptamine in the cortex.     

Inhibition of neuronal uptake reduces the presynaptic effects of clonidine but not of alpha-methylnoradrenaline on the stimulation-evoked release of 3H-noradrenaline from rat occipital cortex slices

The iminoimidazolidine clonidine reduced concentration-dependently the release of 3H-noradrenaline evoked by electrical stimulation from the rate cerebral cortex. Exposure to the neuronal uptake inhibitors cocaine (10 micro M), desipramine (0.1 to 1 micro M) and amphetamine (1 micro M) significantly increased the stimulation-evoked overflow of tritium. These uptake inhibitors antagonized the effects of clonidine on stimulation evoked 3H-noradrenaline release but failed to modify the inhibition induced by the catecholamine alpha-methylnoradrenaline. Inhibition of monoamine oxidase by preincubation of cerebral cortex slices with 0.5 mM pargyline significantly increased the stimulation-evoked overflow of tritium, but clonidine was as effective as in the controls in inhibiting 3H-noradrenaline overflow. The antagonism by desipramine of the clonidine-induced inhibition of neurotransmission could not be attributed to a blockade of presynaptic alpha-adrenoceptors because: (1) the facilitating effect of phentolamine on 3H-noradrenaline overflow was not modified in the presence of desipramine; (2) the magnitude of the inhibition of the stimulation-evoked 3H-noradrenaline release elicited by alpha-methylnoradrenaline was the same in the presence of cocaine or desipramine; (3) exposure to desipramine in the presence of cocaine did not further increase the stimulation-evoked release of 3H-transmitter. Since the catecholamine alpha-methylnoradrenaline inhibited neurotransmission in the presence of desipramine or cocaine, we can conclude that inhibition of neuronal uptake of noradrenaline antagonized selectively the presynaptic inhibitory effects of imidazolines on alpha 2-adrenoceptors. The influence of the inhibition of neuronal uptake on the presynaptic effects of imidazolines and catecholamines should be taken into account when the relative order of potencies of various alpha 2-adrenoceptors agonists is determined.     

Evaluation by reverse phase HPLC of [3H]acetylcholine release evoked from the myenteric plexus of the rat

Summary Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [³H]choline to measure the synthesis and the release of [³H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used.The radiochromatogram following the injection of a [³H]choline-standard and a [¹⁴C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [³H]choline caused the synthesis of [³H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [³H]acetylcholine, whereas the overflow of [³H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mol/l) suppressed the release of [³H]acetylcholine by 60%. Scopolamine (0.1 mol/l) prevented this inhibition and, given alone, enhanced the release of [³H]acetylcholine by 43%. The release of [³H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies.The present experiments show the synthesis and the calcium-dependent release of [³H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats. However, some differences (frequency-dependency of [³H]acetylcholine release, spontaneous neuronal activity) are evident between both species. Reverse phase HPLC is a useful method to separate radioactive choline and acetylcholine with a high recovery rate.Send offprint requests to I. Wessler at the above address     

Cocaine potentiates the responses to methacholine and noradrenaline in the rat vas deferens

The ability of cocaine (10 μM) to potentiate the contractile responses of the epididymal half of the rat vas deferens to methacholine was reversed by prazosin. Prazosin also partially reversed the ability of cocaine to increase the spontaneous overflow of ³ H following loading of the tissue with [³ H]noradrenaline. We suggest that cocaine potentiated the responses to methacholine by stimulating, directly or indirectly, α₁-adrenoceptors.     

Ovarial steroids and methionine-enkephalin modulation of adrenergic transmission in rabbit oviduct

• 1.1. The effect of methionine-enkephalin on the [³H]-noradrenaline ([³H]-NA) overflow and on contractions evoked by field electrical stimulation (FES) and by exogenous NA were studied in vitro in the isthmic part of oviduct of rabbits (untreated and treated with estradiol or progesterone).
• 2.2. The evoked tritium overflow (which reflected [³H]-NA overflow) was determined by liquid scintillation spectrometry.
• 3.3. Field electrical stimulation of 4 Hz (trains of 40 pulses, 0.3 msec) evoked guanethidine-sensitive contractions.
• 4.4. In all groups of animals methionine-enkephalin dose dependently decreased FES-evoked contractions but not those evoked by exogenous NA.
• 5.5. The amount of tritium overflow evoked by 4 Hz stimulation (600 pulses, 1 msec) was significantly lower in tissues from estradiol treated (1.16 ± 0.19%) compared with those obtained in tissues from untreated (1.82 ± 0.22%) and progesterone treated (2.07 ± 0.21%) rabbits. Methionine-enkephalin, 1 μM, decreased the evoked tritium overflow in the isthmus from untreated rabbits by 36.1 ± 3.6%, in estradiol treated by 22.8 ± 2.9% and in progesterone treated by 52.3 ± 4.5%.
• 6.6. The results suggest that the methionine-enkephaline effect on FES-evoked contractions could be due to a prejunctional effect on the adrenergic terminals and that there is a hormonal dependence of the opioid effect on [³H]-NA overflow.

     

Diet-induced atherosclerosis inhibits release of noradrenaline from sympathetic nerves in rabbit arteries

Contractile responses to sympathetic nerve stimulation and exogenous noradrenaline were compared in aortas and pulmonary arteries of control rabbits and rabbits fed a cholesterol-rich diet (0.3%) for 16 or 30 weeks. The diet-induced atherosclerosis reduced the contractions to increasing concentrations of exogenous noradrenaline (0.1 nM to 10 μM) in both arteries, and the reduction was more pronounced after 30 weeks of the hypercholesterolemia. The contractions produced with increasing frequencies of electrical stimulation (1–32 Hz) were nearly abolished in the atherosclerotic arteries. Labeling of the aorta and the pulmonary arteries with [³H]noradrenaline resulted in accumulation of radioactivity in both control and atherosclerotic blood vessels. After mounting the labeled blood vessels for superfusion, a basal efflux of [³H]noradrenaline and of ³H-metabolites was detected. In the atherosclerotic arteries, a decreased efflux of the intraneuronal deaminated metabolites 3,4-dihydroxyphenyl glycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) was detected. Electrical stimulation at 1 Hz (pulmonary artery) or 2 Hz (aorta) caused an augmented efflux of total ³H from the control arteries; this was mostly due to release of intact [³H]noradrenaline. The electrical imp ulses evoked significantly less (16 weeks) or no (30 weeks) release of [³H]noradrenaline in the atherosclerotic arteries. These data illustrate that diet-induced atherosclerosis exerts an inhibitory action on the sympathetic nerve terminals in the aorta and the pulmonary artery of the rabbit. This effect, together with an inhibitory effect at the postjunctional level results in a loss of the responsiveness to nerve stimulation. The atherosclerotic process also inhibits the intraneuronal deamination of the sympathetic transmitter.     

Differences between the neuronal handling of 3H-7- and 3H-7,8-(−)noradrenaline: implications for the release of the labelled neurotransmitter by nerve stimulation

Neighbouring rabbit aortic strips were exposed to a tracer concentration of 3H-7- or 3H-7,8-(-)noradrenaline (bearing 30-35% of its label in position 8) and to 0.5 mumol/l unlabelled (-)noradrenaline for 60 min and then washed in amine-free Krebs solution. Catechol-O-methyl transferase and extraneuronal amine uptake were inhibited throughout. After 114 min of wash-out, the tissue contained less tritium when loaded with 3H-7-(-)noradrenaline than when loaded with 3H-7,8-(-)noradrenaline, and the fractional rate of loss of tritium was greater for the former than for the latter tissues. In the presence of cocaine (to prevent neuronal re-uptake), the same percentage of tissue tritium was released by nerve stimulation (six consecutive periods of stimulation at 1 Hz for 5 min each) in spite of the above difference between tissue tritium levels of the two differently labelled amines. In the absence of cocaine, a higher percentage of tissue tritium was released by nerve stimulation (1 or 3 Hz, 5 min each) for 3H-7- than 3H-7,8-(-)noradrenaline. Unchanged 3H-(-)noradrenaline amounted to 35% of tritium in the stimulation-evoked overflow for 3H-7- and to 50% for 3H-7,8-(-)noradrenaline (frequency of stimulation, 1 Hz). When monoamine oxidase (MAO) was inhibited, no differences were observed between the neuronal handling of 3H-7- and 3H-7,8-(-)noradrenaline, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

α-Adrenoceptor-mediated inhibition of noradrenaline release in rabbit brain cortex slices

Summary Brain cortex slices from rabbits were preincubated with [³H]noradrenaline and then superfused and stimulated electrically at 3Hz. In the presence of cocaine 30 M, unlabelled noradrenaline, -methylnoradrenaline, clonidine, oxymetazoline, xylazine and guanabenz decreased, whereas yohimbine, corynanthine, phentolamine, tolazoline and azapetine increased the stimulation-evoked overflow of tritium. Phenylephrine and prazosin had no effect on the evoked overflow except at concentrations that greatly accelerated the basal outflow of tritium. The results indicate that the noradrenergic axons of rabbit brain cortex are endowed with presynaptic -adrenoceptors which are exclusively of the ₂-type. Addition of various concentrations of cocaine, addition of pargyline, or stimulation at different current strengths was used to obtain either a high or a low stimulation-evoked overflow of tritium. Independently of the method used, a low evoked overflow coincided with a large percentage inhibition produced by 0.1M clonidine, whereas a high evoked overflow coincided with a smaller percentage inhibition produced by clonidine. The results indicate that drugs which block the re-uptake of noradrenaline diminish the presynaptic inhibitory effect of -adrenergic agonists by increasing the biophase concentration of released noradrenaline.     

Intra- and extraluminally-applied acetylcholine on the vascular tone or the response to transmural stimulation in dog isolated mesenteric arteries

Summary Acetylcholine applied extraluminally to isolated, perfused dog mesenteric artery segments produced an endothelium-dependent depressor response when the perfusion pressure was raised by continuous infusion of noradrenaline; the potency was 1/30 to 1/60 that of intraluminal acetylcholine. Contractions induced by transmural electrical stimulation were attenuated by treatment with intra- and extraluminal acetylcholine; the inhibitory effect of intraluminal acetylcholine was greater than that of extraluminal acetylcholine. Removal of endothelium did not significantly alter the inhibitory effect. In mesenteric artery strips with endothelium, treatment with oxyhaemoglobin suppressed the relaxant response to acetylcholine but did not influence the inhibitory effect of acetylcholine on stimulation-evoked contractions. Acetylcholine reduced the ³H-overflow and contraction of superfused mesenteric artery strips, preloaded with ³H-noradrenaline, response to transmural stimulation. By the use of bioassay (dog femoral artery segment with endothelium/coronary artery strip without endothelium), the release of EDRF was first determined in the perfusate, which was introduced to dog mesenteric artery strips loaded with ³H-noradrenaline. The ³H-overflow and contraction caused by the stimulation were not attenuated by EDRF and were also observed following treatment with superoxide dismutase. Inability of the perfusate to reduce the stimulation-evoked ³H-overflow was also observed when the donor and assay tissues were treated with superoxide dismutase. It may be concluded that the inhibition by acetylcholine of the release of neuronal noradrenaline is not dependent on endothelium, Extraluminally applied acetylcholine would reach the endothelium and release EDRF, and intraluminal acetylcholine is presumed to act directly on prejunctional muscarinic receptors; however, acetylcholine appears to cross the medial layer more efficiently from intima to adventitia than in the reverse direction. Send offprint requests to N. Toda at the above address     

Modulation of neural noradrenaline and ATP release by angiotensin II and prostaglandin E2 in guinea-pig vas deferens

Effects of angiotensin II and prostaglandin E₂ on contractions, release of noradrenaline and release of ATP elicited by electrical stimulation (210 pulses, 7 Hz) were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In some experiments postsynaptic a ₁-adrenoceptors and P_2X-purinoceptors were blocked by prazosin and suramin, respectively, to isolate the neural fraction of the overflow of ATP.Electrical stimulation elicited an overflow of tritium and ATP and, in the absence of prazosin and suramin, contraction. In the absence of prazosin and suramin, angiotensin II (1–100 nM) enhanced contractions as well as the evoked overflow of tritium and ATP. All parameters were increased by about the same percentage for a given concentration of angiotensin 11. The effect of prostaglandin E₂ (1–100 nM) was complex. Contractions were mainly enhanced, the evoked overflow of tritium was reduced, whereas the evoked overflow of ATP was predominantly increased. No or almost no contraction remained in the presence of prazosin and suramin, and the evoked overflow of ATP was decreased to about 16%. Angiotensin II (1–100 nM) again enhanced the evoked overflow of tritium and ATP. Both were increased by about the same percentage for a given concentration of angiotensin II and also were increased by about the same percentage as obtained in the absence of prazosin and suramin. Prostaglandin E₂ (1–100 nM) decreased the evoked overflow of tritium and ATP in the presence of prazosin and suramin, both by about the same percentage at a given prostaglandin E₂ concentration.It is concluded that neural release of ATP, like the release of noradrenaline, is presynaptically facilitated by angiotensin II and depressed by prostaglandin E₂. In the case of angiotensin II, increases in neural and postsynaptic ATP release contribute to the increase in ATP over flow observed in the absence of prazosin and suramin. In the case of prostaglandin E₂, an increase in postsynaptic ATP release can override the reduction in neural ATP release and give rise to an increase in ATP overflow in the absence of prazosin and suramin. No evidence for a differential modulation of neural noradrenaline versus ATP release was found. Correspondence to: B. Driessen at the above address     

Prejunctional Muscarinic Receptor Modulation of Noradrenaline Release from Sympathetic Neurones in Rabbit Aorta *

The prejunctional muscarinic modulation of stimulation‐evoked release of ³H‐noradrenaline from sympathetic neurones in rabbit aorta was examined. The role of transmitter uptake, α‐adrenoceptor blockade, stimulation frequency and endothelium on the modulation was investigated. Rings of aorta were incubated with (‐)‐³H‐noradrenaline and subsequently subjected to electrical‐field stimulation. Fractional ³H‐overflow was determined by liquid scintillation counting. Acetylcholine (10^?8–3×10^?6 M) added cumulatively, reduced the stimulation‐evoked ³H‐overflow up to 80%. The effect of acetylcholine was the same in intact and endothelium‐free aorta. The inhibitory effect of acetylcholine was inversely related to the frequency of stimulation (1–10 Hz). The maximal inhibition (%) was 80 (1 Hz), 53 (3 Hz) and 14 (10 Hz). The inhibitory effect of acetylcholine (10^?6 M) and carbachol (10^?5 M) reached a maximum 15 min. after addition and then remained almost constant. Cocaine (3×10^?5 M) did not alter the effect of acetylcholine. Desipramine (10^?6 M) and corticosterone (4×10^?5 M) attenuated the inhibition seen with low concentrations (10^?8–10^?7 M) of acetylcholine. The acetylcholine‐induced inhibition was antagonized by desipramine. Cocaine plus corticosterone attenuated the inhibition seen with high concentrations (10^?6–3×10^?6 M) of acetylcholine. Rauwolscine (10^?6 M) enhanced the maximal inhibitory effect of acetylcholine. We conclude that the inhibitory effect of acetylcholine on ³H‐overflow from rabbit aorta preloaded with ³H‐noradrenaline is (1) inversely related to stimulation frequency; (2) independent of endothelium; (3) unaffected by neuronal and extraneuronal transmitter uptake; (4) that cocaine is not a prejunctional muscarinic antagonist; (5) that cocaine, but not desipramine, is suited as a neuronal uptake inhibitor in studies of prejunctional muscarinic receptor subtypes; and (6) and that there is an inverse interaction between prejunctional α₂‐adrenoceptors and muscarinic receptors.