Monoamine oxidase of types A and B in the saphenous vein and mesenteric artery of the dog

Summary Homogenates of dog saphenous vein and mesenteric artery were prepared in phosphate buffer. Monoamine oxidase (MAO) activity was determined with ³H-5-hydroxytryptamine (³H-5-HT) as a preferential substrate for MAO type A, ¹⁴C--phenylethylamine as a preferential substrate for MAO type B, and ³H-tyramine as a substrate for both MAO types. K _m and V _max for the different substrates were determined, and clorgyline and (–)-deprenyl were used as specific inhibitors. The endogenous noradrenaline content was compared with the activity of MAO in both blood vessels. The results show that the enzymatic deamination of tyramine is slightly but significantly higher in the mesenteric artery than in the saphenous vein. MAO A activity was significantly higher in the mesenteric artery than in the saphenous vein, but MAO B activity was the same in both vessels. Hence, the ratio MAO A activity/MAO B activity was greater for the mesenteric artery than for the saphenous vein. This difference may be related to the density of the adrenergic innervation of the two blood vessels.Some of the results were presented to the 10^th Annual Meeting of the Portuguese Pharmacological Society (Oeiras, 1979) and to the 4^th Meeting on Adrenergic Mechanisms (Porto, 1980)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

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)     

Effects of a monoamine oxidase A inhibitor,FLA 668 (+) on the adrenergic mechanisms of the dog saphenous vein

Summary FLA 668(+)[(S)-(+)-4-amino-2, -dimethylethylphenethylamine (+)-hydrogen bitartrate] selectively inhibited MAO type A in homogenates of dog saphenous vein, with a IC₅₀ of 1 mol/l for MAO A and >1 mmol/l for MAO B.At concentrations of 1 mol/l and higher, FLA 668(+) progressively decreased the formation of deaminated metabolites by saphenous vein strips incubated with³H-noradrenaline, with a preferential action on DOPEG formation. Normetanephrine formation increased but this increase did not wholly compensate for the reduction in the formation of deaminated metabolites.FLA 668(+) caused increased reactivity of saphenous vein strips to noradrenaline and markedly reduced the accumulation of³H-noradrenaline in the incubated tissue; both effects were still evident in the presence of clorgyline. After cocaine, FLA 668(+) caused no further increase in sensitivity.It is concluded that FLA 668(+) is, in the saphenous vein of the dog, a selective inhibitor of MAO type A and that it exerts a cocaine-like effect.Part of the results were presented to the Amine Oxidases: a Cambridge workshop (Caramona et al. 1984). Supported by INIC (Centro de Farmacologia e Biopatologia Quimica da Universidade to Porto)with a grant from the Portuguese-Spanish Academic Exchange Service     

The role played by the extraneuronal system in the disposition of noradrenaline and adrenaline in vessels

Summary The role played by extraneuronal sites in the disposition of noradrenaline and adrenaline was studied in the saphenous vein and in the mesenteric artery of the dog, taking as parameters the influence of blockade of these sites on the sensitivity to and on the time for half-relaxation (t ₅₀) (both in oil and in Krebs solution) of these agonists. Preliminary experiments have shown that the t ₅₀ values are not significantly changed by the changes in the height of the contraction provided the contraction is caused by the same concentration of the agonist.The results obtained permit us to conclude that in both vessels the removal of amines depends on the concentrations used. In low (0.023 and 0.23 M) or in moderately high (2.3 M) concentrations, adrenaline is removed preferentially by extraneuronal sites, whereas noradrenaline preferred neuronal sites.The selectivity of adrenaline for extraneuronal sites was present for such low concentrations that a possible physiological role of these sites in the inactivation of circulating adrenaline must be considered.The results obtained by studying the relaxation in oil or in Krebs solution and by using cortexone (60 M) or U-0521 (dihydroxy-2-methyl propiophenone; 0.1 mM) support the view that, at least in the vein, adrenaline may accumulate in extraneuronal cells and diffuse back into the biophase during the relaxation, thereby slowing the latter.Both in the veins and in the arteries noradrenaline was inactivated more rapidly than adrenaline. The difference in the rate of inactivation of these amines, already observed in controls (when all inactivation pathways are operative) became more marked when both neuronal and extraneuronal sites were blocked. The existence of an important pathway not blocked by cocaine + cortexone + iproniazid which may preferentially inactivate noradrenaline cannot be ruled out.This study was supported by a grant from the Instituto de Alta Cultura (PMC-2)Preliminary results were presented at the VI International Congress of Pharmacology (Helsinki, July 1975)     

Cocaine-sensitive O-methylation of noradrenaline in dental pulp of the rabbit: Comparison with the rabbit ear artery

Summary Incisor pulp from the rabbit metabolises exogenous noradrenaline in concentrations between 0.12 and 1.2 mol/l mainly to NMN.Effects of chronic sympathetic denervation indicated that in incisor pulp the NMN is extraneuronal in origin, and that DOPEG and DOMA formation, as well as a major part of the noradrenaline which accumulates in the tissue, are associated with the sympathetic nerves.NMN formation was unaffected by hydrocortisone 210 mol/l, but was strongly inhibited by cocaine 30 mol/l. These effects contrasted with those in the rabbit ear artery, where NMN formation was increased by cocaine 30 mol/l and decreased by hydrocortisone 210 mol/l.In COMT-inhibited denervated pulp, cocaine inhibited the accumulation of noradrenaline.Monoamine fluorescence histochemistry of pulp exposed to noradrenaline 50 mol/l indicated that cocaine-sensitive uptake occurred in fibroblasts.It is concluded that O-methylation of noradrenaline in dental pulp involves prior uptake of the amine by a process resembling uptake, but which is distinguished from uptake₁ by its extraneuronal location.Abbreviations DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - MAO monoamine oxidase - COMT catecholO-methyl transferase Send offprint requests to I. S. de la Lande at the above address     

Evidence for an extraneuronal location of monoamine oxidase in renal tissues

Summary (1) Homogenates of renal cortex and renal medulla of control and 6-hydroxydopamine-denervated cat kidneys were prepared. (2) Monoamine oxidase (MAO) activity was determined with [³H]-5-hydroxytryptamine ([³H]-5HT) and [¹⁴C]--phenylethylamine ([¹⁴C]--PEA) as preferential substrates for MAO-A and MAO-B, respectively. (3) The endogenous dopamine and noradrenaline tissue contents of control and chemicallydenervated kidneys were compared with the MAO activities. (4) The results show that a 70% depletion of monoamine content by chemical denervation resulted only in a 23% reduction of MAO-A activity in the renal cortex, whereas MAO-13 was unaffected either in the cortical or the medullary zones; in the renal medulla MAO-A activity was not changed by denervation. Most of the MAO activity in the cat kidney is of the B type (74%) and is located in the renal cortex.On leave from Faculdade de Farmácia, Universidade de Coimbra, Portugal Send offprint requests to P. Soares-da-Silva at the above address     

Altered responsiveness of saphenous vein grafts to norepinephrine and tyramine: relation to tissue catecholamine stores.

This study was designed to investigate early changes in reactivity in relation to the adrenergic innervation of venous grafts. Saphenous vein grafts were implanted into the carotid artery by the end-to-end technique in mongrel dogs. After 1 week, the grafts were harvested and dose-response curves to norepinephrine and tyramine were determined and compared with those of nongrafted saphenous veins and carotid arteries. Tissue norepinephrine levels of the blood vessels were measured by HPLC with electrochemical detection. Grafted vessels demonstrated an enhanced sensitivity to norepinephrine and a significant attenuation to tyramine. Additionally, grafted saphenous veins exhibited a significant depletion of norepinephrine content when compared to nongrafted veins. These differences suggest that denervation of the saphenous vein produces a supersensitivity to catecholamines that could account for enhanced vascular reactivity observed following implantation of saphenous vein grafts.     

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

Summary After incubation with 2.3 M 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 M) 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 M) 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 M) 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 -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 -adrenoceptor-mediated positive feedback mechanism in this tissue.     

Predominance of oxidative deamination in the metabolism of exogenous noradrenaline by the normal and chemically denervated human uterine artery

Summary Longitudinal strips were prepared from human uterine arteries obtained at hysterectomy. The artery had a low content of noradrenaline and dopamine, contrasting with a high content of the deaminated catechols, dihydroxyphenylglycol (DOPEG) and dihydroxymandelic acid (DOMA), which together represented 98% of endogenous catechols.When incubated with ³H-noradrenaline (0.1 mol/l), the uterine artery removed, accumulated and metabolized noradrenaline. Deaminated metabolites predominated, DOMA being the most abundant metabolite.Cocaine markedly reduced the accumulation of ³H-noradrenaline and abolished ³H-DOPEG formation, but did not change ³H-DOMA. Selective monoamine oxidase (MAO) inhibitors (clorgyline, selegiline and 2-amino ethyl carboxamide derivatives) caused a marked decrease in the amounts of ³H-DOPEG, ³H-DOMA and ³H-O-methylated and deaminated metabolites (OMDA) formed by the tissue and an increase in ³H-normetanephrine (NMN) formation. Inhibition of catechol-O-methyltransferase suppressed NMN formation and reduced that of OMDA; hydrocortisone slightly depressed the formation of DOMA and OMDA.Homogenates of the uterine artery deaminated ³H-5-HT, ¹⁴C-phenylethylamine and ³H-tyramine; inhibition curves of the deamination of ³H-tyramine by clorgyline and selegiline were compatible with the presence of both MOA A and MOA B.Exposure of the strips to 6-hydroxydopamine (1.5 mmol/l for 20 min; 3 exposure periods followed by washout periods of 15,15 and 30 min) resulted in complete and selective chemical denervation of the arterial tissue. This chemical denervation had effects which were similar to those of cocaine. The 2-amino ethyl carboxamide derivatives markedly reduced the formation of deaminated metabolites by the denervated strips.The semicarbazide-sensitive amine oxidase inhibitor semicarbazide reduced the formation of ³H-DOMA and ³H-DOPEG in intact strips, but was devoid of action in the denervated ones.It is concluded that, in the human uterine artery, deamination predominates over O-methylation and that extraneuronal deamination, leading to the formation of DOMA (and of OMDA) plays a major role in the metabolism even of low concentrations of exogenous noradrenaline.Abbreviations COMT Catechol O-methyltransferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - HPLC-ED high pressure liquid chromatography with electrochemical detection - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase - NMN normetanephrine - 6-OHDA 6-hydroxydopamine - OMDA O-methylated and deaminated metabolites of noradrenaline (3-methoxy-4-hydroxyphenylglycol and 3-methoxy-4-hydroxymandelic acid) - Ro 01-2812 3,5-dinitropyrocatechol - Ro-19-6327 N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide hydrochloride - Ro 41-1049 N-(2-aminoethyl)-5-(mfluorophenyl)-4-thiazole carboxamide hydrochloride Supported by Instituto Nacional de Investigação Científica (INIC, FmP1) and Junta Nacional de Investigação Científica e Tecnológica (JNICT). Fatima Martel is a PhD student with a grant from JNICT Send offprint requests to W. Osswald at the above address     

Phorbol 12,13-dibutyrate,an activator of protein kinase C,stimulates both contraction and Ca2+ fluxes in dog saphenous vein

Summary The vascular effects of phorbol 12,13-dibutyrate (PDBu) were studied in the dog saphenous vein. PDBu (1 M) caused contraction (0.58 ± 0.22 g/mg wet wt.) and Ca uptake (74.2 ± 41.2 mol/kg wet wt.) which were unaffected by 10 M phentolamine (N = 6). The PDBu-induced contraction was greatly (60–80%) inhibited in Ca²⁺-free solution. 15 Ca efflux measurements performed in Ca²⁺-free solution showed that PDBu did not cause Ca release from intracellular storage sites. The contractile response to PDBu (1 nM-1 M) was significantly correlated with the magnitude of Ca uptake; contraction and the rise in tissue Ca²⁺ also had a similar time course. Correlation between the two measures persisted when the responses to PDBu were augmented by co-administration with 20 mM KCl. However, no synergism occurred between the two agonists. Both the contraction and Ca uptake responses to PDBu were reduced by nifedipine and verapamil, each at 1 M. In the Triton X-100 skinned saphenous vein, where the voltage-dependent Ca channel is not functional, 10 M PDBu did not cause contractions in the presence of 0.1 M Ca²⁺. Thus, contraction of the intact saphenous vein by PDBu characteristically exhibits great Ca dependence and PDBu seems to activate the voltage-dependent Ca channel, presumably through stimulation of protein kinase C; the ensuing Ca entry is primarily responsible for contraction. However, the mechanism responsible for the PDBu-induced contractions that are resistant to Ca²⁺-free PSS or Ca entry blockers remains to be defined. It appears that the dog saphenous vein differs from dog femoral artery, rabbit aorta and pig carotid artery where PDBu contractions do not display dependence on external Ca²⁺. Send offprint requests to P. J. S. Chin at the above address     

Purine agonists prevent trophic changes caused by sympathetic denervation

Surgical denervation of the lateral saphenous vein of the dog causes marked extraneuronal changes, both of a morphological and functional type. In an attempt to investigate the factor(s) responsible for the trophic effects exerted by the sympathetic innervation on the dog saphenous vein we studied the effects of noradrenaline, adenosine, inosine and N-ethylcarboxamidoadenosine (NECA) on vascular tissue after sympathetic denervation. The saphenous vein was denervated using either surgical or chemical (6-hydroxydopamine, 6-OHDA) methods. Noradrenaline (0.1 microgram/kg per h), adenosine (10 micrograms/kg per h), inosine (10 micrograms/kg per h) or NECA (0.1 microgram/kg per h) were delivered continuously for 5 days through Alzet minipumps connected to the vein. 6-OHDA-induced denervation resulted in morphological changes similar to those described for surgical denervation. Smooth muscle cells and fibroblasts showed ultrastructural signs of increased synthetic activity and their size was significantly increased. In confirmation of earlier studies, constant i.v. infusions of noradrenaline did not prevent the morphological changes induced by denervation. Adenosine prevented the morphological changes induced by chemical or surgical denervation. Similarly to adenosine, infused NECA prevented the structural consequences of denervation. In contrast, inosine did not prevent the changes caused by surgical denervation. The results are compatible with an involvement of purines in the trophic effects of sympathetic innervation. Moreover, the effects of adenosine do not appear to be mediated by inosine.     

Functional activity of the noradrenergic innervation of large cerebral arteries.

1 The role of the sympathetic innervation of cerebral arteries remains controversial. Therefore, the functional activity of the adrenergic innervation of the rabbit basilar artery was characterized and compared to that of a peripheral artery, the ear artery. 2 Both the ear artery and basilar artery have similar endogenous noradrenaline (NA) contents but accumulation of [3H]-NA was considerably greater in the basilar artery. 3 Studies of tritium efflux after loading with [3H]-NA demonstrated a considerable non-neuronal component since neither guanethidine nor tetrodotoxin completely blocked tritium efflux during nerve stimulation. Pretreatment with blockers of uptake2 did not eliminate this problem. 4 Comparison of methods for estimating the functional activity of adrenergic nerves showed that, for the vessels studied, NA content and [3H]-NA accumulation gave markedly different answers. Fractional release of [3H]-NA did not correspond to fractional release of endogenous NA. 5 Adrenergic nerves innervating cerebral arteries are shown to have a high activity relative to a peripheral artery. While cerebrovascular sympathetic innervation may not play an important role in normal circumstances, its influence may be seen in pathological conditions.     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

Facilitatory presynaptic angiotensin receptors on the sympathetic nerves of the human saphenous vein and pulmonary artery. Potential involvement in β-adrenoceptor-mediated facilitation of noradrenaline release

Summary Spirally cut strips of human saphenous vein and pulmonary artery preincubated with ³H-noradrenaline were superfused in the presence of corticosterone and desipramine or cocaine. In the saphenous vein angiotensin I, angiotensin II and angiotensin III concentration-dependently increased the electrically (2 Hz) evoked tritium overflow (relative order of potency: angiotensin II > angiotensin I > angiotensin III). The angiotensin receptor antagonist saralasin displaced the concentration-response curve of angiotensin II to the right, and also blocked the facilitatory effect of angiotensin III. Captopril, an inhibitor of angiotensin converting enzyme, did not modify the concentration-response curve of angiotensin I and did not significantly diminish the release-increasing effect of the nonselective -adrenoceptor agonist isoprenaline, whereas saralasin attenuated the facilitatory effect of the ₂-adrenoceptor agonist procaterol. In the pulmonary artery the angiotensin receptor agonist Val⁵-angiotensin II-Asp¹--amide also increased the electrically evoked tritium overflow in a concentration-dependent manner. It is concluded that the sympathetic nerve fibres of the human saphenous vein (and probably of the human pulmonary artery as well) are endowed with facilitatory presynaptic angiotensin receptors. Angiotensin I exerted its facilitatory effect in the saphenous vein probably via direct stimulation of angiotensin receptors but not by conversion to angiotensin II. Furthermore, the ₂-adrenoceptor-induced facilitation of noradrenaline release may in part be mediated by local stimulation of angiotensin II synthesis, which may occur by increased formation or activation of renin and/or increased availability of angiotensinogen.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert at the above address     

Effects of angiotensin I and angiotensin II in blood vessels: greater influence of converting enzyme activity in the rabbit basilar artery

We investigated the constrictor effects of Angiotensin I (Ang I) and Angiotensin II (Ang II) on rabbit peripheral (aorta, carotid artery, mesenteric artery, saphenous artery) and cerebral (basilar artery) vessels and in rat aorta in functional organ bath studies. The effect of angiotensin converting enzyme (ACE) inhibition by captopril was also assessed in these preparations. Ang II elicited concentration-dependent contractions with comparable potency in rabbit and rat endothelium-free vascular rings (pD₂ about 8.5) which indicates a lack of species and regional variation in the contractile responses to Ang II. The responses to Ang II were reduced by the presence of a functional endothelium in rabbit mesenteric artery and in rat aorta. Since ACE determines the plasma and tissue conversion of Ang I to active Ang II, we calculated the ratio R (EC₅₀ Ang I-induced contraction: EC₅₀ Ang II-induced contraction) as an indicator of the tissue ACE effectiveness. In the aorta without endothelium, Ang I was found to be much less potent than Ang II in the rabbit (R = 44) compared with the rat (R = 3.5). This species difference in the aortic conversion of Ang I to Ang II was confirmed by the use of captopril. Captopril (10^–6M) shifted the Ang I concentration/response curve by 2- and 14-fold to the right in rabbit and rat respectively. In other rabbit blood vessels, the rank order of potency to Ang I in endothelium denuded rings was basilar artery carotid artery aorta saphenous artery. In addition, the R value was the lowest for the basilar artery (R = 2.5). This is in agreement with the highest rightward shift (78-fold) of the Ang I concentration/response curve by captopril for basilar artery in comparison with only 3-, 8- and 3-fold shifts observed in carotid artery, saphenous artery and aorta respectively. In conclusion, our data provide evidence for a greater influence of ACE in rabbit basilar artery than in peripheral vessels.     

Effects of clorgyline and (−)deprenyl on the deamination of normetanephrine and noradrenaline in strips and homogenates of the canine saphenous vein

Summary The influence of specific inhibitors of MAO A (clorgyline) and MAO B [(–)deprenyl] on the metabolism of normetanephrine (NMN), in strips of canine saphenous vein was studied, both in the absence and in the presence of inhibitors of neuronal (cocaine) and extraneuronal (hydrocortisone) uptake. Moreover, the formation of metabolites of noradrenaline and of NMN by saphenous vein homogenates and the influence of clorgyline or (–)deprenyl on this formation are described.Clorgyline reduced to the same degree (by about 70%) the formation of methoxy-hydroxy-phenylglycol (MOPEG) and of vanillylmandelic acid (VMA) in strips incubated with NMN, whereas (–)deprenyl reduced by about 50% the formation of MOPEG and had no effect on VMA production. Hydrocortisone had effects very similar to those of (–)deprenyl.Saphenous vein, homogenates ()-methylation inhibited), deaminated both noradrenaline and NMN; clorgyline and (–)deprenyl reduced the formation of metabolites of both noradrenaline and NMN.It is concluded that both MAO A and B are able to deaminate noradrenaline and NMN, but that in the intact tissue the former has no access to MAO B. Even in intact tissues MAO B may play a role in the metabolism (but not in the inactivation) of noradrenaline by deaminating the NMN formed from noradrenaline and giving preferentially origin to MOPEG.Supported by Instituto Nacional de Investigação Científica (INIC, FmPl)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

The effects of drugs and denervation on removal and accumulation of noradrenaline in the perfused hind-limb of the dog

Summary The removal of noradrenaline by the autoperfused hind-limb of dogs anaesthetized with pentobarbital, as well as the accumulation of noradrenaline in the saphenous vein were studied. Sensitivity of the perfused vascular area was determined by the response of the perfusion pressure to infusions of noradrenaline.The removal of noradrenaline declined very slowly during infusions lasting for up to 2 h, but edema of the perfused limb occurred after 45 to 60 min; therefore, the duration of infusion was limited to 30 min. During this period, noradrenaline was infused in rates increasing by a factor of 2 and ranging from 0.5 to 16 g/kg per minute. Accumulation capacity was saturated at 1 g/kg · min^–1, but the amount removed increased until a four-to eightfold rate was reached and then levelled off.At a rate of 1 g/kg · min^–1 the influence of drugs and of surgical denervation was investigated in other experimental series. Cocaine, nialamide, phenoxybenzamine and pretreatment with reserpine reduced removal (by 50, 45, 40 and 35%, respectively). Cortexone had no detectable influence on removal with this rate of infusion, but blocked it effectively when 4 g/kg · min^–1 were infused. Accumulation of noradrenaline in the vein was prevented by cocaine or reserpine, slightly reduced by phenoxybenzamine and enhanced by nialamide. The effects of nialamide plus cocaine did not differ significantly from those of cocaine alone, but cortexone plus cocaine completely blocked removal and accumulation. Surgical denervation reduced removal by about 70% and abolished accumulation; reserpine plus nialamide had similar effects. In the case of nialamide, removal progressively diminished during the infusion period and this time dependence of effects was accompanied by a prolongation of noradrenaline washout.Cocaine, reserpine and denervation caused supersensitivity of the perfused vessels to noradrenaline, whereas nialamide and cortexone had no such effect and phenoxybenzamine caused subsensitivity.The pronounced ability of the perfused vessels of the hind-limb to remove noradrenaline from the circulating blood is attributed primarily to neuronal uptake and intraneuronal oxidative deamination; extraneuronal uptake and inactivation seem to play an important role when neuronal mechanisms are saturated (infusion of higher noradrenaline doses) or impaired (after cocaine or denervation).Supported by Instituto de Alta Cultura (Research Project PMC/2). Part of this work was presented at the Fifth International Congress on Pharmacology (S.Francisco, July 23–28, 1972).     

The actions of 5-hydroxytryptamine receptor agonists and antagonists at pre- and postjunctional level on the canine saphenous vein

Summary The prejunctional and postjunctional 5-HT receptors of the canine saphenous vein were studied. The release of ³H-noradrenaline (³H-NA) from incubated saphenous vein strips was inhibited by 5-hydroxytryptamine (5-HT) in a concentration-dependent way (5-HT concentrations: 0.01, 0.1 and 1.0 mol · l^–1), but not by the selective 5-HT_1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 1 and 10 mol · l^–1). The inhibitory effect of 5-HT was antagonized by metitepine and methysergide, but not by yohimbine, (–)-pindolol or ketanserin. In strips preincubated with 5-HT (1.2 mol · l^–1), the fractional release of ³H-NA was slightly reduced (paired experiments). 5-HT and 8-OH-DPAT caused concentration-dependent contractions of the saphenous smooth muscle. A parallel shift of the concentration-response curve for 8-OH-DPAT to the right was caused by metitepine and yohimbine, but not by ketanserin. The contractions caused by 5-HT were antagonized by metitepine and yohimbine (parallel displacement of the curves to the right), as well as by ketanserin and methysergide (with a depression of the upper part of the curve). Blockade of -adrenoceptors (due to prazosin plus a low concentration of yohimbine) also resulted in a weak antagonistic effect. Ketanserin and metitepine displaced the noradrenaline concentration-response curve to the right. We conclude that the saphenous vein of the dog is endowed with prejunctional receptors of the 5-HT₁ type which can not be classified as belonging either to the 1 A or 1 B subtype; and that at the postjunctional level 5-HT₁ (possibly of the 1D subtype) and 5-HT₂ receptors are present. 5-HT is able to activate all these receptors.Supported by INIC (Centro de Farmacologia e Biopatologia Química da Universidade do Porto) Send offprint requests to M. Q. Paiva at the above address     

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     

Different characteristics of postjunctional alpha-adrenoceptors on arterial and venous smooth muscle

Summary The postsynaptic alpha-adrenoceptors of rabbit ear artery, dog splenic artery and dog splenic vein were characterized in vitro by the use of the selective antagonists prazosin (alpha₁) and rauwolscine (alpha₂). In isolated segments of rabbit aar artery, prazosin was a potent antagonist of norepinephrine-induced contraction, with a receptor dissociation constant (K_B) of 6.4 nM. In contrast, rauwol-scine was effective only at very high concentrations (K_B=4,000 nM), suggesting that the alpha-receptor of the ear artery has conventional alpha₁-characteristics. The opposite order of potency was observed in a presynaptic alpha-receptor model, the isolated guinea pig atrium, where rauwolscine was potent (K_B=4.5 nM) and prazosin essentially inactive.Rauwolscine had an intermediate potency in the dog splenic vasculature, with a K_B of 125 nM in splenic arterial segments and 5–15 nM in segments or helical strips of splenic vein. In contrast to the results with rauwolscine, prazosin was essentially equipotent in dog and rabbit tissues as an antagonist of norepinephrine-induced contraction (K_B=3–9 nM). Schild analysis showed competitive antagonism for both rauwolscine and prazosin in all tissues. These results are consistent with the presence of a homogenous population of alpha-receptors in dog vasculature having characteristics of both the alpha₁ and alpha₂ receptor subtypes.