Localization of monoamine oxidase of type A and B in blood vessels with different innervation patterns

Summary Homogenates of dog mesenteric artery, dog saphenous vein, denervated saphenous vein, rabbit ear artery, denervated ear artery and human umbilical artery were prepared in phosphate buffer. Monoamine oxidase activity was determined with [³H]-5-hydroxytryptamine ([³H]-5-HT) as a preferential substrate for MAO type A and [¹⁴C]--phenylethylamine as a preferential substrate for MAO type B. The endogenous noradrenaline content was compared with the MAO activities in these blood vessels. The results show that there is a good relationship of MAO type A content with the density of adrenergic innervation, but it is evident that this type of MAO is present even in vessels devoid of adrenergic innervation. Thus, MAO type A in these vascular structures has both intraneuronal and extraneuronal locations whereas MAO type B is predominantly or exclusively extraneuronal.Some of the results were presented to the 11th Annual Meeting of the Portuguese Pharmacology Society (Lisboa, 1980) and to the 8th International Congress on Pharmacology (Tokyo, 1981)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

The effect of corticosterone on extraneuronal amine uptake and effector response in rat salivary glands

The effect of corticosterone on effector cell response to noradrenaline in vivo and on extraneuronal amine uptake in vitro has been investigated in rat submaxillary glands. When tissue slices were incubated with [³H]noradrenaline the level of extraneuronally retained radioactive material was found to be markedly reduced at a concentration of 10 μg ml^?1 of corticosterone after inhibition of neuronal uptake by protriptyline. Corticosterone in a dose of 10 mg kg^?1 was found to markedly potentiate the secretory response to noradrenaline in vivo, when the neuronal uptake of noradrenaline was blocked by protriptyline (10 mg kg^?1, i.p.). Inhibition of neuronal uptake alone by protriptyline or of the extraneuronal uptake alone by corticosterone in the doses used here did not affect the dose-response curve for noradrenaline, at least not in its lower part. The data thus clearly show that the extraneuronal amine uptake of rat salivary glands is blocked by corticosterone and that this extraneuronal uptake might be regarded as a mechanism of importance for the inactivation of the adrenergic transmitter.     

Effects of S-11701 on accumulation, release and metabolism of norepinephrine in isolated canine saphenous veins.

1. The effects S-11701 ([morpholinyl-2)-methoxy]-8-tetrahydro-1,2,3,4 quinoline) on accumulation, overflow and metabolism of [3H]norepinephrine were investigated in isolated canine saphenous veins. 2. Saphenous veins were incubated with [3H]norepinephrine in the absence or the presence of S-11701; the drug caused a concentration-dependent inhibition of the tissue content of [3H]norepinephrine and its metabolites, except for 3-methoxy-4-hydroxymandelic acid (VMA). 3. In helical strips of canine saphenous veins previously incubated with [3H]norepinephrine and then suspended for isometric tension recording and measurement of the overflow of labelled transmitter and its metabolites, S-11701 (30 microM) significantly increased the spontaneous efflux of total 3H; this effect was almost exclusively due to an augmentation of the efflux of [3H]DOPEG. 4. During electrical stimulation (9 V, 1 Hz), S-11701 at 1 microM slightly increased the overflow of extraneuronal norepinephrine metabolites without affecting the contractile response. At the higher concentration (30 microM) the compound increased the contractive response and the overflow of 3H; the latter was due mainly to an increase in [3H]DOPEG and, to a lesser extent, in [3H]norepinephrine. 5. DMI (1 microM) did not interfere with the effects of S-11701 on DOPEG efflux. 6. These experiments indicate that in the canine saphenous vein, S-11701 causes a concentration-dependent inhibition of neuronal accumulation of [3H]norepinephrine. At higher concentrations, S-11701 enters the adrenergic nerve terminals independently of the neuronal amine carrier and displaces [3H]norepinephrine from its storage sites.     

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.     

Some mechanisms which may modulate noradrenaline release in guinea-pig isolated ileum

Guinea-pig isolated ileum pre-incubated with [³ H]noradrenaline, released ³ H-label during coaxial stimulation at 3·2-12·8 Hz. This evoked ³ H-overflow seems due to nerve stimulation, and may be frequency-dependent. The ³ H-overflow evoked by coaxial stimulation was increased with 2·6 μM phentolamine, reduced at threshold stimulation by 0·56 μM prostaglandin E₁, but the tendency for 2·8 μM indomethacin to increase the evoked ³ H-overflow was not statistically significant. The results indicate that the release of noradrenaline from guinea-pig ileum may be modulated by activation of presynaptic α-adrenoceptors, whereas prostaglandins act mainly at a postsynaptic site to modulate the sympathetic response.     

Modulation of the concentration of noradrenaline at the neuro-effector junction in human saphenous vein.

We studied the importance of neuronal and extraneuronal uptake and of the pre-junctional alpha-adrenergic feed-back mechanism for the junctional noradrenaline concentration in the human saphenous vein. All major metabolites of the enzymatic breakdown of noradrenaline were detected in the overflow of superfused veins loaded with [3H]-noradrenaline. The efflux of 3,4-dihydroxyphenylglycol (DOPEG) was drastically reduced in preparations labelled after neuronal uptake blockade indicating its neuronal origin; the other metabolites are formed extraneuronally since they behaved distinctly differently from DOPEG under several experimental conditions. Extraneuronal uptake followed by enzymatic breakdown removes the same amount of noradrenaline from the biophase during nerve activity as that diffusing intact out of the tissue, whereas neuronal uptake appears only half as effective since the overflow of intact noradrenaline increases by only 48% in the presence of desmethylimipramine (DMI). However, in preparations mounted for isometric tension recording, neuronal uptake blockade potentiated contractions to alpha-adrenergic activation, emphasizing the functional importance of the neuronal disposition mechanism. By contrast, no evidence was found for a hydrocortisone-sensitive extraneuronal uptake compartment, suggesting that extraneuronal removal may have little, if any, functional importance. During nerve stimulation, yohimbine increased the amount of labelled noradrenaline present in the superfusate, while exogenously added noradrenaline decreased it in the presence of cocaine. Thus, prejunctional alpha-adrenoceptors can modulate the junctional concentration of neurotransmitter in the human saphenous vein.     

Prejunctional alpha 2-adrenoceptors modulate stimulation-evoked norepinephrine release in rabbit lateral saphenous vein

Segments of rabbit lateral saphenous vein prelabelled with [3H]noradrenaline were perfused and superfused with physiological salt solution. Tritium overflow evoked by transmural nerve stimulation (3 Hz for 2 min) was abolished by tetrodotoxin (1 microM). The selective alpha 2-adrenoceptor agonist UK 14,304 inhibited stimulation-evoked 3H-overflow in a concentration-dependent manner, with an IC50 of 71 nM. In contrast, the alpha 2-adrenoceptor agonist B-HT 933 had no effect on 3H-overflow in concentrations up to 10 microM. The selective alpha 2-adrenoceptor antagonists idazoxan and SKF 86466, as well as the non-selective alpha-antagonist phentolamine, facilitated the nerve stimulation evoked 3H-overflow, with an order of potency of idazoxan greater than or equal to phentolamine greater than SK&F 86466. Prazosin (100 nM) had little effect on 3H-overflow. These findings suggest that stimulation-evoked neurotransmitter release is modulated via prejunctional alpha 2-adrenoceptors.     

Variation in noradrenaline output with changes in stimulus frequency and train length: Role of different noradrenaline pools

1 During adrenergic nerve stimulation the output/pulse of noradrenaline from the rabbit vas deferens and portal vein is not constant but increases as the stimulus frequency or train length is increased. Depending upon the stimulus frequency and train length the fractional release of noradrenaline may vary from less than 10^-7 to greater than 10^-4.

2 Endogenous tissue stores of noradrenaline were labelled by incubation with (-)-[³H]-noradrenaline and [¹⁴C]-tyrosine. The output/pulse of newly synthesized [¹⁴C]-noradrenaline remained constant as the train length was increased whilst the output/pulse of [³H]-noradrenaline increased under the same conditions. This phenomenon was independent of the stimulus frequency. Newly synthesized noradrenaline also appeared in the superfusate following nerve stimulation more rapidly than exogenously loaded noradrenaline.

3 Both [³H]-noradrenaline and [¹⁴C]-tyrosine were found to label an easily releasable store of noradrenaline. Mobilization from this store was the same at low and high frequencies of nerve stimulation.

4 It is concluded that at least two functional stores of noradrenaline exist within the adrenergic nerve ending. Newly synthesized noradrenaline is probably only a minor constituent of transmitter output under normal conditions of adrenergic nerve activity.

5 At least two mechanisms control the amount of noradrenaline released per pulse during nerve stimulation. Facilitation of release with increasing train lengths appears to be due to the mobilization of transmitter from a secondary store. Facilitation of release with increasing stimulus frequency is not dependent on mobilization from any particular store and at present there is no satisfactory explanation for this phenomenon.

     

The recovery of the capacity for uptake-retention of ( 3 H)noradrenaline in rat adrenergic nerves after reserpine

The uptake retention of [³H]noradrenaline (2.5 μg/kg, i.v., 30 min before death) in rat salivary glands was studied at different times after reserpine treatment (10 mg/kg, i.p.). The effect of removing the cervical superior ganglion 12 h before death on the recovery of the [³H]noradrenaline uptake-retention capacity after reserpine was also investigated. The ganglionectomy was unilateral, and the contralateral side was always preganglionically denervated. In glands with uninterrupted postganglionic adrenergic nerves the onset of recovery of the [³H]noradrenaline retention capacity occurred 24–36 h after reserpine. Normal contents were found on the second to third days. Between day 3 and 6 a possible overshoot of [³H]nor- adrenaline content, followed by normal and subnormal contents (7–21 days) were recorded. Ganglionectomy, 12 h before death, markedly delayed the recovery of [³H]noradrenaline retention capacity. Both the recovery curve for [³H]noradrenaline retention in glands with intact postganglionic nerves, and the effect of ganglionectomy on the [³H]noradrenaline retention capacity, were clearly related to the relative number of new functioning amine granules that are transported via the axons to the nerve terminals at different times after the reserpine-pretreatment. The results indicate that young amine granules, recently transported to the nerve terminals via the axons, have the greatest capacity to take up and retain [³H]noradrenaline. The half-life of this capacity in the young granules appears to be about 12 h. Since published results indicate that [³H]noradrenaline is initially taken up in the “small easily releasable pool” of transmitter, we suggest that young amine granules are of the greatest importance for adrenergic function, i.e. that they are particularly active in taking up recaptured noradrenaline, in the synthesis, and in the release of this transmitter.     

Simultaneous measurement of [3H]noradrenaline release and neurogenic contraction under identical conditions, to determine the prejunctional inhibitory effects of SKF 99101H and BRL 56905 in dog saphenous vein

Using a tissue bath system which allowed the simultaneous measurement of electrically-induced [³H]nor-adrenaline release and neurogenic contraction under identical conditions, we investigated the prejunctional inhibitory activity of the selective 5-HT_1D/1B receptor agonists BRL 56905 ((±)-3-amino-6-carboxamido-1,2,3,4-tetrahydrocarbazole) and SKF 99101H (3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate), compared to sumatriptan and 5-HT. Transmural electrical stimulation (2 Hz) of dog saphenous vein induced consistent increases in [³H]noradrena- line release as well as reproducible contractile responses (<10% decrease over four stimulation periods). BRL 56905, SKF 99101H, sumatriptan and 5-HT (60 nM – 6 μM) inhibited electrically-evoked [³H]noradrenaline release and neurogenic contractile responses in dog saphenous vein. However, despite being measured under identical conditions, the inhibition of [³H]noradrenaline release was consistently greater than the inhibition of neurogenic contraction induced by a particular concentration of agonist, suggesting that neurogenic contractile responses in dog saphenous vein result from the combined release of noradrenaline and other non-noradrenergic neurotransmitters. Under the present assay conditions, since the agonists produced only small (BRL 56905, sumatriptan and 5-HT) or marginal (SKF 99101H) contractile responses, it is unlikely that this is the cause of the discrepancy observed between inhibition of release and inhibition of contraction. The inhibitory effects of BRL 56905, sumatriptan and 5-HT were blocked by the 5-HT_1D/1B receptor antagonist methiothepin, consistent with the involvement of canine ca-5-HT_1D/1B receptors in inhibiting neurotransmitter release and subsequent smooth muscle contraction in dog saphenous vein. The present results show that the novel 5-HT_1D/1B receptor agonists BRL 56905 and SKF 99101H are at least as potent as sumatriptan and 5-HT, at activating prejunctional inhibitory ca-5-HT_1D/1B heteroreceptors on sympathetic axon terminals in dog saphenous vein. In addition, when measured simultaneously in the same tissue preparation, [³H]noradrenaline release was inhibited to a much greater extent than neurogenic contraction by any particular agonist. Received: 7 October 1996 / Accepted: 23 December 1996     

Subcellular distribution of [3H]amphetamine and [3H]guanethidine and their interaction with adrenergic neurons

The subcellular distribution of [³H]amphetamine and [³H]guanethidine and their interaction with each other and with noradrenaline binding sites have been examined. The ratio p/(p + s) × 100, an indication of affinity for noradrenaline storage particles, for [³H]amphetamine and [³H]guanethidine was 12% and 57% respectively. Protriptyline, a substance which inhibits amine transport mechanism at the level of the cell membrane, i.e. the membrane pump, and reserpine, an agent which impairs incorporation of amines into the storage particles in the adrenergic nerve fibre, inhibited the uptake and storage respectively, of [³H]guanethidine more than that of [³H]amphetamine. Retention of [³H]guanethidine by rat salivary glands was markedly decreased by sympathetic denervation of the glands while that of [³H]amphetamine was not. The results suggest that guanethidine possesses a much higher affinity for noradrenaline binding sites than amphetamine.     

An investigation of the role of adrenergic innervation in the regulation of the extraneuronal uptake of [3H]-isoprenaline into rat vasa deferentia and atria.

Destruction of adrenergic nerves by neonatal guanethidine treatment did not affect the extraneuronal accumulation of [3H]-isoprenaline by adult vasa deferentia or atria. Neither pre- nor post-ganglionic denervation of vasa deferentia resulted in a significant change in extraneuronal accumulation of [3H]-isoprenaline. The appearance and subsequent development of extraneuronal uptake in embryonic and newborn rats did not appear to be dependent on a fully developed or functional adrenergic innervation. Decreasing plasma catecholamine levels by adrenal demedullation did not have an effect on extraneuronal uptake. Chronic cold exposure (7 days) significantly increased plasma levels of adrenaline, and there was a significant decrease in the corticosterone-sensitive extraneuronal uptake of [3H]-isoprenaline into atria. After 14 days in the cold, plasma adrenaline levels had fallen, and were no longer significantly different from control levels. The extraneuronal uptake of [3H]-isoprenaline into atria after 14 days cold exposure was no longer different from that of control atria. There did not appear to be a direct correlation between plasma catecholamines and extraneuronal accumulation of [3H]-isoprenaline. Neither the presence of adrenergic nerves nor plasma catecholamines appear to play a major role in the regulation of extraneuronal uptake by atria and vasa deferentia of the rat.     

Calcium dependence of prejunctional inhibitory effects of adenosine and acetylcholine on adrenergic neurotransmission in canine saphenous veins

In canine blood vessels acetylcholine and adenosine inhibit the exocytotic release of norepinephrine during nerve stimulation. The present experiments were designed to determine the Ca2+ dependence of these prejunctional effects. Segments of canine saphenous veins were mounted for isometric tension recording in organ chambers filled with Krebs-Ringer or Tyrode solution. Altering the Ca2+ concentration of the solution did not affect the inhibitory response to acetylcholine during nerve stimulation; the prejunctional potency of adenosine was inversely related to the Ca2+ concentration of the bath content. the ionophore A23187 caused contractions which were inhibited by phentolamine, verapamil, and adenosine but were augmented by acetylcholine. Helical strips of dog saphenous veins were incubated in [3H]norepinephrine and mounted for superfusion and determination of [3H]norepinephrine in the superfusate. A23187 increased the overflow of [3H]norepinephrine. Acetylcholine augmented this efflux; by contrast adenosine decreased the release induced by the ionophore. The results demonstrate that the prejunctional effect of acetylcholine was not due to direct interference with the availability of Ca2+ for the electro-secretory process in adrenergic nerve terminals and suggest that adenosine interferes either with the coupling role of the activator ion or its extrusion from the neuroplasm.     

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     

Reverse trans-synaptic regulation of neuronal noradrenaline uptake.

It was established that the blocking agent of β-adrenoceptors, propranolol (5.10^?7–1.10^?6 M), activates [³H]noradrenaline uptake by isolated rat organs (vas deferens, spleen, small intestine, atrium, uterus) by 30–180 per cent. The blocking agent of α-adrenoceptors, phentolamine (5.10^?7–1.10^?6M), activates noradrenaline uptake by 30–55 per cent only in the organs possessing postsynaptic α-adrenoceptors (vas deferens, spleen, small intestine). The activator of β-adrenoceptors, isopropylnoradrenaline (5.10^?7–1.10^?6M), was shown to produce a decrease in [³H]noradrenaline uptake by 15–50 per cent in all the organs investigated. The substances activating a-adrenoceptors—urea (2.10^?3–5.10^?3M) and mesatone (1.10^?6–1.10^?5M)—inhibit [³H)noradrenaline uptake in the organs with postsynaptic α-adrenoceptors by 20–45 per cent. Activation of neuronal [³H]noradrenaline uptake induced by phentolamine (1.10^?6M) is due to the release of a humoral factor from the effector cell and its influence on adrenergic neurone. Mesatone (1.10^?5M) causes the formation and release of a humoral factor inhibiting neuronal [³H]noradrenaline uptake. The formation of humoral factors changing the intensity of amine uptake is related to the activation of protein synthesis in the effector cell. The possible mechanism of the reverse trans-synaptic regulation of neuronal noradrenaline uptake via the adrenoceptors of the effector cell is discussed.     

Effects of hypoxia on noradrenaline release and neuronal reuptake in isolated rabbit thoracic aortic strips

Summary To clarify the effects of hypoxia on stimulus-evoked noradrenaline release and on neuronal reuptake of the released noradrenaline, we examined the effects of hypoxia on contraction responses of rabbit thoracic aortic strips to transmural electrical stimulation and on the stimulation-evoked overflow of total [³H] and [³H]noradrenaline from the strips prelabelled with [³H]noradrenaline. This was done in the presence or absence of an inhibitor of neuronal uptake (cocaine). In a medium equilibrated with a gas mixture of 95% O₂/5% CO₂ (control), cocaine doubled the stimulation-evoked overflow of total [³H] and [³H]noradrenaline; there was a concomitant increase (130%) in contractions to electrical stimulation. At 0% O₂ (95% N₂/5% CO₂, hypoxia), cocaine had no significant effects on either the stimulation-evoked overflow of total [³H] and [³H]noradrenaline or contractions. In the absence of the drug, hypoxia decreased the stimulation-evoked overflow of total [³H] and [³H]noradrenaline to 47% and 43%, respectively, of the control values, whereas these values were 31% and 28%, respectively, after exposure to cocaine. The inhibition by hypoxia of contraction responses to electrical stimulation was greater in the presence of cocaine than in its absence. These results show that hypoxia inhibits both noradrenaline release evoked by a given stimulus and neuronal uptake.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 offprint requests to S. Miwa at the above address     

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.     

Pharmacological characterization of presynaptic α-adrenoceptors modulating [3H]noradrenaline and [3H]5-hydroxytryptamine release from slices of the hippocampus of the rat

The experiments served to characterize the receptors mediating the inhibitory effect of α-adrenergic drugs on K⁺ (20 mM)-induced [³H]noradrenaline (NA) and [³H]5-hydroxytryptamine ([³H]5-HT) release from slices of the dorsal part of rat hippocampus. Dose-response curves were constructed using the cumulative dose-response technique (Frankhuyzen and Mulder, 1982). All of the adrenergic agonist drugs examined inhibited the K⁺-induced [³H]NA release. NA appeared to have the highest intrinsic activity followed by adrenaline. Clonidine and adrenaline had similar intrinsic activities, while that of oxymetazoline was lowest. The highest pD₂ values were observed for oxymetazoline and clonidine, being slightly higher than that of adrenaline followed by NA. By far the lowest pD₂ values was observed for phenylephrine. With the exception of phenylephrine, all of the agonists also inhibited the K⁺-induced [³H]5-HT release. NA, adrenaline and oxymetazoline appeared to have similar intrinsic activities, while that of clonidine was considerably lower. The pD₂ values of NA and adrenaline were not significantly different but were somewhat lower than those of oxymetazoline and clonidine. Similar antagonistic effects of phentolamine and yohimbine were observed with respect to the adrenergic inhibition of K⁺-induced [³H]NA and [³H]5-HT release. Prazosin, however, appeared to be ineffective in both instances. It is concluded from these results that the presynaptic adrenergic inhibition of [³H]NA as well as [³H]5-HT release is mediated by α₂-adrenoceptors located on noradrenergic and serotonergic varicosities, respectively. Furthermore, our data suggest that these α₂-adrenoceptors are not pharmacologically identical.     

Presynaptic changes promoted by alloxan diabetes in the cat isolated heart

Adrenergic presynaptic functions were evaluated in the cat isolated perfused heart preparation. The sympathetic nerve endings were labelled with [3H]noradrenaline ([3H]NA) and the effect of electric neural stimulation was determined in the presence of drugs which inhibit neuronal or extraneuronal uptake, or which antagonize alpha-adrenoceptors. [3H]NA overflow was measured in control and diabetic cats and was significantly increased by electric neural stimulation on both conditions. Perfusion with 0.1 microM phentolamine increased transmitter overflow in control hearts but failed to do so on organs obtained from alloxan-treated cats. The data provide evidence that in alloxan diabetic cats there is an abnormality of the adrenergic synapse.     

Structural and functional alterations caused at the extraneuronal level by sympathetic denervation of blood vessels

Summary The lateral saphenous vein of the dog and the rabbit ear artery were surgically denervated, by clamping the vessel or by removal of the superior cervical ganglion, respectively. Both procedures resulted in denervation of the vessels.The denervated, lateral saphenous vein was supersensitive to exogenous noradrenaline and inactivation of the amine (in oil immersion experiments) was slower in denervated vein strips than in control strips treated with cocaine. Incubation experiments with ³H-noradrenaline confirmed that denervated strips formed considerably fewer metabolites than control ones (in the absence or presence of cocaine) and that O-methylation of noradrenaline was reduced by about 50%. When the strips were incubated with ³H-isoprenaline, the denervated ones accumulated and metabolized isoprenaline to a lesser degree than control strips. Hydrocortisone did not reduce the accumulation of isoprenaline in the denervated vein and had only minor effects on O-methylation. The metabolism of noradrenaline and isoprenaline gradually recovered with time.In the ear artery, denervation was accompanied by a marked reduction in O-methylation, but not in accumulation, of isoprenaline. In both vessels there was a highly significant positive correlation between noradrenaline content and O-methylating capacity; in the saphenous vein accumulation of isoprenaline was also positively correlated to noradrenaline content.Morphological changes observed in the denervated vessels consisted essentially in dedifferentiation of smooth muscle cells (which attained larger dimensions, had an indented, large nucleus, augmented euchromatin and an increased amount of ribosomes), abundance of extracellular material and fibroblasts. Mast cells were present in denervated veins (but not in controls) and the histamine content was increased in the former. Structural alterations were homogeneously distributed in the saphenous vein but restricted to the adventitio-medial area in the rabbit ear artery. Depletion of endogenous noradrenaline by reserpine pretreatment did not cause the alterations seen after denervation. On the other hand, continuous intravenous infusion of noradrenaline during 5 days did not prevent, and even worsened, the alterations caused by denervation. It was concluded that noradrenaline does not appear to be the factor the lack of which is exclusively responsible for the impairment of the extraneuronal system, in the denervated tissue.Taken together, the data show that the sympathetic innervation of blood vessels exerts a regulatory function on extraneuronal events; the disappearance of innervation results in marked impairment of the corticosteroid-sensitive O-methylating system and in morphological changes of both smooth muscle cells and fibroblasts.Part of the results were presented at 3rd Joint Meeting of the French and Spanish Pharmacological Societies in Toulouse, 1981 (Branco et al. 1982)