Serafim Guimarães

Adrenergic receptors in the veins of the dog

Abstract The actions of catecholamines and adrenergic blocking drugs on the veins of the dog (posterior vena cava, portal vein, femoral vein, lateral saphenous vein) were studied in vitro . In these experiments quantitative and qualitative differences were found. Superficial veins were more sensitive. Adrenaline and dopamine had direct actions causing contractions blocked by phenoxybenzamine (pM50-6.40) and erotamine (pA2-8.20). Isoprenaline caused relaxation and/or contraction depending on the vein, tone and dose; relaxation was blocked by pronethalol (pA2-6.51) and contractions were antagonized or reversed by ergotamine and phenoxybenzamine. Evidence for the existence of both alpha and beta receptors in the veins, the former prevailing, is presented. Ergotamine induced marked venoconstriction, at least in part through alpha receptor activation; no evidence for a beta blocking action of ergotamine was found. The results in vivo (autoperfused femoral vein, vena cava, and small veins of the paw) correlate well with those found in vitro . Agonists and antagonists exerted effects which can be attributed to the presence of alpha and beta adrenergic receptors. However, in these experiments isoprenaline never caused activation of alpha receptors, probably because the doses used were much lower than those used in vitro .

A study of the adrenoceptor-mediated feedback mechanisms by using adrenaline as a false transmitter

SummaryAfter incubation with 2.3 μM 3H-(±)-adrenaline(3H-AD) or 3H-(-)-noradrenaline(3H-NA) for 60 min in the presence of hydrocortisone and U-0521, dog saphenous vein strips were perifused (the fluid containing cocaine+hydrocortisone+U-0521) and electrically stimulated. Tritium fractional release per shock was calculated for 1 and 5 Hz.Phentolamine (3 μM) enhanced the overflow of tritium evoked by electrical stimulation at both frequencies but at 1 Hz the enhancement was higher for strips loaded with 3H-AD than for strips loaded with 3H-NA.Propranolol (1 μM) reduced the overflow evoked by electrical stimulation at 1 Hz from the strips loaded with 3H-AD but not from those loaded with 3H-NA.Isoprenaline (0.04 μM) increased the overflow of tritium evoked by electrical stimulation at 1 Hz from the strips loaded with 3H-NA but did not change that from strips loaded with 3H-AD.It is concluded that: a) the α-adrenoceptor-mediated feedback mechanism is also present in the dog saphenous vein; b) this feedback mechanism also functions with the “false transmitter” AD; c) the use of 3H-AD as false transmitter revealed the existence of a β-adrenoceptor-mediated positive feedback mechanism in this tissue.

Further study of the adrenoceptors of the saphenous vein of the dog: influence of factors which interfere with the concentrations of agonists at the receptor level.

In the present study the affinities of some sympathomimetic amines for alpha- and beta-adrenoceptors of the dog saphenous vein tissue were determined after all known factors interfering with the concentration of these agonists at the receptor level had been assessed and excluded. It was observed that in control experiments the relative potencies of sympathomimetic agonists for inducing contractions were: adrenaline (1.6) greater than noradrenaline (1.0) greater than phenylephrine (0.38) greater than isoprenaline (0.009). The elimination of neuronal uptake by cocaine, 4 X 10(-6) M, enhanced predominantly the effects of noradrenaline (by a factor of 7.5), whereas block of catechol-O-methyl transferase (COMT) by U-0521, 10(-4) M, only enhanced those of adrenaline (by a factor of 2.6) and block of beta-adrenoceptors by propranolol, 5 X 10(-7) M, enhanced those of isoprenaline (by a factor of 3) and adrenaline (by a factor of 1.8). Block of COMT enhanced the effects of adrenaline approximately as much as did the blockade of neuronal uptake; this seems to indicate that the affinity of adrenaline for extraneuronal and neuronal uptake processes is approximately the same. Regarding the relaxation-inducing capacity of sympathomimetic agents it was observed that isoprenaline, adrenaline and noradrenaline are full agonists, whereas phenylephrine was not able to produce relaxation amounting to more than 5% of the maximum. Denervation did not modify the relaxant effects of isoprenaline. After elimination of all known factors interfering with the concentration of the sympathomimetic agonists in the biophase, the ratios between the ED50s of each amine for alpha- and beta-adrenoceptors were: adrenaline = 34, noradrenaline = 109 and isoprenaline = 0.0041.

Relation between the amount of smooth muscle of venous tissue and the degree of supersensitivity to isoprenaline caused by inhibition of catechol-O-methyl transferase.

SummaryThe relation between the smooth muscle cell mass of dog saphenous vein strips and the degree of supersensitivity to isoprenaline caused by U-0521 (3,4-dihydroxy-2-methyl propiophenone), an inhibitor of the catechol-O-methyl transferase (COMT), was studied. For the quantitative determination of smooth muscle mass, the thickness of the muscle layer as determined by light microscopy and the maximal shortening induced by supramaximal concentration of phenylephrine were used.After the strips had been contracted by 3×10−6 M phenylephrine, a concentration which was able to produce an about 90% maximal contraction, dose-response curves to the relaxant effect of isoprenaline were determined in the absence and in the presence of U-0521 (10−4 M).It was observed that U-0521 caused marked supersensitivity to the relaxant effect of isoprenaline (varying between 3 and 81 times), as well as an increase of the maximal relaxation caused by this amine (varying between 7 and 120%). The correlation between these data and the smooth muscle cell mass shows that there was a direct proportionality between these parameters.Oxytetracycline (10−4 M), an inhibitor of binding of catecholamines to collagen, did not produce any enhancement of the effects of isoprenaline.It is concluded that COMT is related to smooth muscle cells in this tissue.

The effects of cocaine and denervation on the sensitivity to noradrenaline, its uptake and the termination of its action in isolated venous tissue.

SummaryThe sensitivity to noradrenaline and the termination of its action in the isolated venous tissue of the dog as well as the capacity of the tissue to remove noradrenaline from the incubation medium were studied either in normal, surgically denervated or cocaine-treated strips. Cocaine or denervation had strikingly similar effects on the parameters under study, causing supersensitivity and inhibition of noradrenaline inactivation and uptake mechanisms; in no instance did cocaine differ significantly from denervation. However, both cocaine and denervation exerted a more marked influence on sensitivity to than on inactivation of noradrenaline. These results suggest that neuronal uptake plays roles of different importance in regulating the initial concentration of noradrenaline at the receptor level and in terminating the action of noradrenaline, probably because it is the first mechanism to operate when the strip is exposed to noradrenaline but has a relatively low capacity.Venous strips had a considerable capacity for removing noradrenaline from the incubation medium. More than half the removed noradrenaline was accumulated in neuronal structures; the rest seems to have been extraneuronally metabolized. Excellent correlations between noradrenaline content and removal capacity, sensitivity to noradrenaline, inactivation capacity and potentiation by cocaine were found. On the whole the results give evidence for a prejunctional site of action of cocaine.

Alpha2-adrenoceptor-mediated responses to so-called selective alpha1-adrenoceptor agonists after partial blockade of alpha1-adrenoceptors

SummaryIn dog saphenous vein — a tissue possessing both postsynaptic α1- and α2-adrenoceptors — the effects of two selective α1-adrenoceptor agonists (phenylephrine and methoxamine) were compared with that of the selective α2-adrenoceptor agonist, UK-14,304, before and after phenoxybenzamine. Furthermore, the influence exerted by prazosin, yohimbine and verapamil on the effects of these agonists was also studied before and after phenoxybenzamine. In the absence of phenoxybenzamine, prazosin (56 nmol/l) caused a parallel shift of the concentration-response curves of both phenylephrine and methoxamine to the right (by 0.94 and 1.1 log units, respectively) and had no effect on the concentration-response curve of UK-14,304, while 20 nmol/l yohimbine caused a marked parallel shift of the concentration-response curve of UK-14,304 to the right (by 1.18 log units) and caused only minor displacements of those of phenylephrine and methoxamine (by 0.2 and 0.33 log units, respectively). After exposure of the strips to 30 nmol/l phenoxybenzamine, prazosin (56 nmol/l) caused small shifts of the concentration-response curves of both phenylephrine (by 0.36 log units) and methoxamine (by 0.31 log units) and did not change that of UK-14,304, while yohimbine (20 nmol/l) caused pronounced parallel shifts of the concentration-response curves (to the right) of all the agonists: phenylephrine (by 1.0 log units), methoxamine (by 0.93 log units) and UK-14,304 (by 1.28 tog units). When UK-14,304 was added to the bath during a sub-maximal contraction to phenylephrine it caused a further contraction almost up to the maximum; if this procedure was repeated after phenoxybenzamine (30 nmol/1), there was no further contraction to UK-14,304.In the absence of phenoxybenzamine, verapamil (5 μmol/l) caused a parallel shift of the concentration-response curve of phenylephrine (or methoxamine) to the right and a non-parallel shift (with marked depression of the maximal effect) of that of UK-14,304. However, after phenoxybenzamine (30 nmol/l), the same concentration of verapamil caused non-parallel shifts of the concentration-response curves of the three agonists to the right with about equal depression of the maximal effects. We conclude that, after removal of α1-adrenoceptor reserve by phenoxybenzamine, the responses to selective α1-adrenoceptor agonists are predominantly α2-adrenoceptor-mediated. This may explain why under these conditions, the selective α1-and α2-adrenoceptor agonists are equally antagonized by calcium entry blockers.

Differential distribution in, and release from, sympathetic nerve endings of endogenous noradrenaline and recently incorporated catecholamines

SummaryGuinea-pig vasa deferentia or hypogastric nerve-vas deferens preparations, preincubated with pargyline (to irreversibly inhibit monoamine oxidase), were exposed to 2.3 μmol/l of unlabelled adrenaline or of 3H-7-(−)-noradrenaline in the presence of hydrocortisone (to inhibit extraneuronal uptake). The vasa deferentia were then mounted in perifusion chambers and subjected to transmural electrical stimulation, electrical stimulation of the nerve, depolarization by potassium (50 mmol/l), or addition of tyramine (40 μmol/l). The evoked overflow of tritium and of unlabelled catecholamines was expressed as a fraction of their tissue content. For all stimuli, the fractional release of the exogenous amines was higher than that of endogenous noradrenaline. Thus, recently incorporated amines are preferentially mobilized irrespective of the particular type of releasing mechanism or the chemical nature of the amine.In vasa deferentia which had been loaded with increasing amounts of adrenaline (by incubating the tissues with adrenaline at concentrations ranging from 0.6 to 160 μmol/1), the fractional release of adrenaline decreased and became closer to that of endogenous noradrenaline. Hence, the access of exogenous catecholamines to the deepest storage sites requires higher concentrations of amines than those needed to reach the more easily releasable pools.Light microscope autoradiographs obtained from slices of vasa deferentia previously loaded with 2.3 μmol/l 3H-(−)-noradrenaline showed that the outer layers were strongly labelled with silver grains whereas the inner layers were poorly marked. It is concluded that recently incorporated amines are preferentially stored in varicosities close to the surface of the tissue and, in comparison with endogenous noradrenaline, are preferentially released from sympathetically innervated organs.

The role of neuronal and extraneuronal systems in the metabolism of adrenaline and noradrenaline released from nerve terminals by electrical stimulation

SummaryStrips of dog saphenous vein were preincubated with 1.4 μM 3H-(±)-adrenaline or 3H-(−)-noradrenaline and then perifused and electrically stimulated. The effects of some drugs on the metabolism of the released amine was studied through the estimation of the different 3H-compounds appearing both in the spontaneous outflow and in the overflow evoked by electrical stimulation. a)Spontaneous Outflow. Both for noradrenaline and adrenaline the major part of the tritium efflux in controls was represented by metabolites: deaminated metabolites predominated for noradrenaline and O-methylated ones for adrenaline. 3,4-dihydroxy-2-methyl propiophenone (U-0521; 100μM), cocaine (8.8 μM) and hydrocortisone (41 μM) did not cause major changes in the metabolite pattern. However, iproniazid (180 mg/kg, i.v.) reduced the efflux of 3,4-dihydroxyphenylglycol (DOPEG) and of O-methylated deaminated metabolites (OMDA) and increased that of metanephrine (MN) (or normetanephrine-NMN) and of the unchanged amine. The results are compatible with the view that the metabolites appearing in the spontaneous outflow are formed predominantly presynaptically.b)Overflow Evoked by Electrical Stimulation. Both for adrenaline and noradrenaline cocaine practically abolished the formation of DOPEG, while U-0521 abolished the formation of MN (or NMN). The formation of DOPEG, abolished by cocaine, reappeared in the presence of cocaine + U-0521 and was again diminished when hydrocortisone was added to this combination; thus, both noradrenaline and adrenaline, released from the nerves by electrical stimulation, can be deaminated extraneuronally. Cocaine did not reduce either OMDA or MN (or NMN); this supports the view that O-methylation occurs predominantly or exclusively in extraneuronal sites. Inhibition of MAO, much more than inhibition of COMT, increased the amount of intact noradrenaline appearing in the overflow, the opposite occurring for adrenaline. On the other hand, for adrenaline, inhibition of MAO caused an increase of MN concomitant with a corresponding decrease of OMDA; and for noradrenaline, inhibition of COMT caused an increase of DOPEG, concomitant with a corresponding decrease of OMDA. Thus, we conclude that for adrenaline, OMDA is predominantly formed from MN by subsequent deamination, while for noradrenaline, OMDA is predominantly formed from DOPEG by subsequent O-methylation.

The role of angiotensin II in hypertension due to adenosine receptors blockade.

The renin-angiotensin system may be involved in hypertension induced by adenosine receptors blockade with 1,3-dipropyl-8-sulfophenylxanthine (DPSPX). Contractions of the mesenteric vasculature to angiotensin II, noradrenaline and potassium chloride were studied in DPSPX-induced hypertension. Male Wistar rats received infusions of saline or DPSPX (90 microg kg(-1) h(-1), i.p.) for 3 or 7 days. Blood pressure was determined by the tail-cuff method. On days 3 or 14, concentration-response curves were obtained on mesenteric arteries and veins. Plasma angiotensin II levels, measured by radioimmunoassay, were higher in DPSPX-hypertensive rats. The maximum contractile effect of angiotensin II was lower in vessels from DPSPX-hypertensive rats while that for noradrenaline was higher. Potassium chloride-induced contractions were larger in veins from DPSPX-hypertensive rats but similar in arteries, when compared with control rats. We conclude that raised angiotensin II levels and altered vascular reactivity are consistent with a renin-angiotensin-mediated hypertension.

Uptake inhibitors do not change the effect of imidazoline α2-adrenoceptor agonists on transmitter release evoked by single pulse stimulation in mouse vas deferens

SummaryThe present study was undertaken to compare the presynaptic interaction of neuronal noradrenaline uptake inhibitors with imidazoline and phenylethylamine α2adrenoceptor agonists under two different conditions: at low and high noradrenaline concentrations in the biophase.Isolated mouse vasa deferentia were stimulated with trains of 7 pulses given at 0.2 Hz and the inhibition by the α2-adrenoceptor agonists clonidine, α-methylnoradrenaline, and UK-14,304 of twitch responses was measured in the absence and in the presence of either cocaine (12 μmol/l) or desipramine (40 nmol/l). The effects were determined for the first (equivalent to single pulse stimulation) and the last stimulus of each train. Both uptake inhibitors antagonized the presynaptic inhibitory effects of imidazolines (clonidine and UK-14,304) on the last twitch; the effects on the first twitch remained unchanged. In contrast, the uptake inhibitors potentiated the inhibitory effect of the phenylethylamine (α-methylnoradrenaline) on both the first and the last twitches.These results support the view that the concentration of noradrenaline in the biophase plays a decisive role in the inhibition by a2-adrenoceptor agonists of the electrically evoked release of noradrenaline. Agonists that are not substrates of neuronal uptake (i.e., clonidine, UK-14,304) become less effective when noradrenaline is present in the biophase while substrates of neuronal uptake (i. e., α-methylnoradrenaline) do not. The results argue against the hypothesis that uptake inhibitors interact directly with presynaptic α2-adrenoceptors or act at some link between uptake and receptor sites.