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)     

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.     

Superoxide dismutase partially prevents sympathetic denervation by 6-hydroxydopamine

Summary The effects of superoxide dismutase (S.O.D.) in two models of chemical denervation induced by 6-hydroxydopamine (6-OHDA) were studied.To evaluate the effects of S.O.D. on in vitro 6-OHDA-induced denervation, fragments of the lateral saphenous veins of mongrel dogs were pre-incubated in oxygenated Krebs-Henseleit solution with or without S.O.D. and then incubated under control conditions, with 6-OHDA or with 6-OHDA + S.O.D. Following the incubation period the fragments were repeatedly washed with Krebs solution and then used for determination of noradrenaline and for morphological study. 6-OHDA produced a profound depletion of noradrenaline. This depletion was significantly reduced although not prevented by S.O.D. The protective effect of S.O.D. was concentration-dependent. The ultrastructural study confirmed the 6-OHDA-induced sympathetic nerve degeneration as well as the protective effect afforded by S.O.D.In order to evaluate the effects of S.O.D. on in vivo 6-OHDA-induced denervation, male Wistar rats were anaesthetized and the tail vein cannulated. Saline or S.O.D. were intravenously delivered. 6-OHDA was injected five minutes after the beginning of infusions. Fragments of the left ventricle and vasa deferentia were used for determination of noradrenaline and for morphological study. 6-OHDA produced a significant depletion of noradrenaline in the left ventricle and vas deferens (to 8% and 18% of control values respectively). This depletion was reduced, though not prevented by S.O.D. Morphological data confirmed the neurotoxic effect of 6-OHDA and a protective role for S.O.D.In the concentration shown to afford protection against in vitro 6-OHDA-induced denervation, S.O.D. neither chemically inactivated 6-OHDA, nor did it exert any blocking effect on the neuronal uptake of ³H-noradrenaline. Thus, the protection afforded by S.O.D. against chemical denervation by 6-OHDA appears to be due to the free radical scavenging effect of S.O.D. Send offprint requests to A. Albino-Teixeira at the above address     

Effect of cocaine and related drugs on the uptake of noradrenaline by heart and spleen

Noradrenaline uptake by heart and spleen after intravenous infusion of noradrenaline was measured in the pithed rat. Cocaine, given before the infusion, inhibited the noradrenaline uptake in relation (a) to the dose administered and (b) to the amount of noradrenaline infused. There was an association between increase in the pressor response to a test dose of noradrenaline and inhibition of the uptake by the heart. Drugs related chemically to cocaine, such as α-cocaine, amethocaine, and atropine, did not alter the noradrenaline uptake or potentiate the blood pressure response to noradrenaline. The noradrenaline uptake by the heart was unchanged after dibenamine, but blocked by the dichloro-analogue of isoprenaline. It was concluded that cocaine specifically prevented the uptake of noradrenaline by tissues, thus increasing the amount of noradrenaline available for combination with adrenergic receptors. The dichloro-analogue of isoprenaline appeared to block both uptake by the heart and the combination with receptors.     

Potentiation of alpha-adrenoceptor-mediated responses following chronic beta-adrenoceptor stimulation in the rat heart.

1. Noradrenaline and isoprenaline were infused subcutaneously in rats by use of Alzet osmotic minipumps. The effects of catecholamine infusion on ventricular alpha- and beta-adrenoceptor density and also the responses of isolated cardiac tissues were compared with saline infusion. 2. Noradrenaline (1 mg kg-1) or isoprenaline (40 micrograms kg-1) infused for 3 days resulted in a desensitization of beta-adrenoceptor-mediated responses of isolated left atria and papillary muscles. Concentration-response curves to isoprenaline were shifted to the right in left atria whilst maximum responses were reduced in papillary muscles. Right atrial rate responses were not affected by infusions of catecholamines. 3. Infusions of either noradrenaline or isoprenaline resulted in a supersensitivity of alpha-adrenoceptor-mediated responses in isolated papillary muscles with leftward displacements of concentration-response curves to phenylephrine. 4. The density of both ventricular [3H]-dihydroalprenolol and [3H]-prazosin binding sites was reduced following noradrenaline infusion. Isoprenaline infusion reduced only the density of [3H]-dihydroalprenolol binding sites. 5. Noradrenaline infusion therefore 'down-regulates' both alpha- and beta-adrenoceptors in the rat heart but at the same time ventricular alpha-adrenoceptor-mediated responses are enhanced. Isoprenaline similarly enhances responses to phenylephrine and possible mechanisms for this phenomenon are discussed.     

Comparison of activity of alpha-adrenoceptor agonists and antagonists in dog and rabbit saphenous vein

Summary The alpha adrenoceptors present on the saphenous vein of the dog and rabbit were characterised in vitro using the selective alpha₁ antagonist prazosin and the alpha₂ antagonists rauwolscine and yohimbine. In the dog saphenous vein, prazosin and rauwolscine competitively antagonised contractile responses to phenylephrine and UK-14,304 respectively. Noradrenaline was competitively blocked by prazosin only. In contrast, phenylephrine and methoxamine-induced responses in the rabbit saphenous vein were insensitive to prazosin and corynanthine. Rauwolscine competitively blocked responses to UK-14,304, noradrenaline and phenylephrine and pA₂ values were similar against each agonist. Noradrenaline and UK-14,304 were equipotent agonists on the rabbit saphenous vein while in the dog vein noradrenaline has a lower potency than UK-14,304. These results suggest that the dog saphenous vein has a mixed population of alpha adrenoceptors, while the alpha adrenoceptors on the rabbit vein are homogeneous, with characteristics of the alpha₂ subtype.     

Effects of basal and acetylcholine-induced release of endothelium-derived relaxing factor on contraction to alpha-adrenoceptor agonists in a rabbit artery and corresponding veins.

1. The effects of an endothelium-dependent (acetylcholine) and an endothelium-independent (sodium nitroprusside) relaxant against noradrenaline-induced contractions were compared in three isolated superficial blood vessels of the rabbit, the lateral saphenous vein, plantaris vein and distal saphenous artery. Both produced concentration-related relaxations of all three vessels and were more effective against submaximal than maximal contractions to noradrenaline. Transient contractions to high concentration of acetylcholine occurred only in endothelium-intact preparations of saphenous vein and were inhibited by flurbiprofen. 2. In endothelium-denuded preparations sodium nitroprusside was 3 times more effective than in endothelium-intact preparations, while acetylcholine (less than 3 microM) was inactive. Sensitivity was similar for each relaxant: lateral saphenous vein greater than or equal to plantaris vein greater than distal saphenous artery. The similar profile of sodium nitroprusside and acetylcholine suggests that differences in susceptibility to endothelium-derived relaxing factor (EDRF) are caused by inter-vessel variations in the excitation-coupling process for noradrenaline. 3. Haemoglobin inhibited acetylcholine-induced relaxations in the endothelium-intact preparation of the lateral saphenous vein and distal saphenous artery, which suggests a similar EDRF in each preparation and the likelihood that this is a single substance, presumably nitric oxide. 4. The influence of basal, spontaneously released EDRF on alpha-adrenoceptor function was tested either by mechanical disruption of the endothelium or by adding haemoglobin to endothelium-intact segments. Endothelial disruption slightly reduced contractions to noradrenaline (NA) in distal saphenous artery but increased response size of lateral saphenous and plantaris veins, in the latter also increasing sensitivity to NA: haemoglobin mimicked endothelial disruption. Thus, basal release of EDRF like acetylcholine and nitroprusside was more effective in the veins than in the corresponding artery. 5. In lateral saphenous vein responses to phenylephrine were enhanced by endothelial disruption, but without change in sensitivity: responses to UK-14304, B-HT 920 and cirazoline, which had a relatively slow speed of onset of contraction were not affected. There was no correlation between enhancement and alpha-adrenoceptor sub-type although the agonists which were enhanced all activate alpha 1-adrenoceptors. Competitive antagonists failed to reveal an alpha-adrenoceptor subtype enhanced by endothelial disruption. However, effects of phenoxybenzamine suggest that alpha 1-adrenoceptors are necessary for the influence of basal EDRF.     

Changes in brain catecholamine levels following olfactory bulbectomy and the effect of acute and chronic administration of desipramine in rats

Bilateral olfactory bulbectomy of the rat caused marked changes of noradrenaline level in several brain regions accompanied with the development of mouse-killing behavior (muricide). Noradrenaline level increased in the medial amygdala, ventromedial and lateral hypothalamus in muricidal olfactory bulbectomized rats (OB rats) but not in non-muricidal OB rats, while dopamine level decreased in the lateral hypothalamus in muricidal OB rats. Acute administrations of desipramine not only suppressed muricide of OB rats but normalized noradrenaline change in ventromedial hypothalamus and dopamine change in lateral hypothalamus. Chronic administration of desipramine also suppressed muricide and normalized noradrenaline changes in ventromedial and lateral hypothalamus and medial amygdala. These findings suggest that the increase in noradrenaline levels in the medial amygdala, ventromedial and lateral hypothalamus may be important for the induction of muricide in OB rats, and muricide was suppressed by desipramine in accordance with the normalization of increased noradrenaline levels, and that the change in dopaminergic function in the lateral hypothalamus may also be important for this muricide.     

A regional difference in the distribution of postsynaptic alpha-adrenoceptor subtypes in canine veins

The responsiveness of helical venous strips isolated from fifteen different sites in the body of dogs to relatively selective alpha 1- and alpha 2-adrenoceptor agonists was studied, as well as to a non-selective alpha-adrenoceptor agonist. Longitudinal strips of the portal and mesenteric veins and the inferior vena cava between the liver and the renal vein (segment C) were also investigated. All veins contracted to noradrenaline or phenylephrine whereas only seven veins responded significantly to clonidine: the saphenous, cephalic, jugular and femoral veins and longitudinal strips of the portal and mesenteric veins and the segment C of the inferior vena cava. The brachiocephalic, azygos, pulmonary and splenic veins and the superior vena cava and the supradiaphragmatic portion (segment A) and the infrarenal portion (segment D) of the inferior vena cava responded little to clonidine. Unlike the longitudinal strips, the helical strips of the portal and mesenteric veins and the segment C of the inferior vena cava did not respond to clonidine. According to the relative sensitivities to phenylephrine and clonidine, those veins which responded to clonidine could be divided into three groups. (1) The veins in which the sensitivity to phenylephrine was higher than to clonidine: longitudinal strips of the portal vein and segment C of the inferior vena cava, (2) the veins whose sensitivity to phenylephrine was lower than to clonidine: the saphenous, cephalic, femoral and external jugular veins, (3) the vein whose sensitivity to the two agonists was comparable: longitudinal strips of the mesenteric vein. Subtype characteristics were further analyzed in the saphenous vein and in the portal vein using prazosin, phentolamine and yohimbine as antagonists. Analysis of Schild plots to noradrenaline suggested that a mixed population of alpha-adrenoceptor subtypes might be present in the saphenous vein, whereas a rather homogeneous population of a single subtype might occur in the portal vein. The results of the antagonism experiment against phenylephrine and clonidine suggested that contractions of the saphenous vein are mediated by both alpha 1- and alpha 2-adrenoceptors whereas contractions of the portal vein are exerted mainly through alpha 1-adrenoceptors. The results suggest that there may be a distinct regional difference with respect to postsynaptic alpha- adrenoceptor subtypes in the canine venous system.     

Investigation of a combined arteriolar dilator and beta-adrenoceptor antagonist (SK & F 92657) in the peripheral vessels of man.

1 SK & F 92657 is a new compound which in animals has both precapillary vasodilator and beta-adrenoceptor blocking activity. The effects of the drug on forearm blood flow and dorsal hand vein distensibility have been studied in healthy volunteers. 2 Forearm blood flow was measured by venous occlusion plethysmography. The drug was infused directly into a brachial artery. The effects of SK & F 92657 on resting flow and on the dose-response relationship for intra-arterial isoprenaline were studied. 3 Venous distensibility was measured in the large veins on the back of the hand. The drug was infused directly into a selected vein and its effects on resting tone and on the responses to noradrenaline and to isoprenaline were investigated. 4 Intra-arterial infusion of SK & F 92657 over 2 min caused a dose-dependent increase in forearm blood flow which developed gradually to reach a maximum after approximately 40 min. Infusion into dorsal hand veins at 100 ng/min and 500 ng/min had no dilator effect in veins preconstricted with noradrenaline. At higher doses SK & F 92657 potentiated the constrictor effect of noradrenaline. 5 In both veins and arteries SK & F 92657 attenuated the dilator effects of isoprenaline. The drug had no effect on the venodilator effects of bradykinin, histamine or sodium nitroprusside. 6 The pattern of response of these vessels to locally infused SK & F 92657 is exactly that expected of a drug combining the properties of hydralazine-like vasodilatation and beta-adrenoceptor blockade. Furthermore, in the arterioles, both properties were manifest at the same dose.     

Comparison of the effects of desipramine on noradrenaline- and methoxamine-evoked venoconstriction in man.

1. The dorsal hand vein compliance technique was used to investigate the dual effect of tricyclic antidepressants at the noradrenergic synapse (i.e. noradrenaline uptake blockade leading to potentiation and alpha 1-adrenoceptor blockade leading to antagonism of the effect of noradrenaline). The effects of a single oral dose (100 mg) of desipramine (DMI) on venoconstrictor responses to locally infused noradrenaline and methoxamine, a selective alpha 1-adrenoceptor agonist with little affinity for the uptake mechanism, were examined. 2. Eight healthy male volunteers participated in four weekly experimental sessions. Each session was associated with one of the following treatment conditions: noradrenaline/DMI, noradrenaline/placebo, methoxamine/DMI, methoxamine/placebo. Subjects were allocated randomly to treatments and sessions according to a double-blind balanced design. Noradrenaline acid tartrate (0.33-33 ng min-1) and methoxamine hydrochloride (0.0135-135 micrograms min-1) were infused into the superficial dorsal hand vein; each dose was infused for 5-7 min with 5 min intervening washout periods. Systolic and diastolic blood pressure and pulse rate were recorded before the infusion and immediately after the infusion of the highest dose. Salivation, an index of anticholinergic activity of the antidepressant, was measured by the dental roll technique. 3. Both noradrenaline and methoxamine produced dose-dependent venoconstriction: the geometric mean ED50 for noradrenaline was 4.41 ng min-1 and for methoxamine was 2558 ng min-1; the potency ratio (noradrenaline/methoxamine) was 2884. DMI shifted the dose-response curve for noradrenaline to the left (ANOVA: P < 0.025), resulting in a dose-ratio of 0.26. DMI did not affect the dose-response curve for methoxamine significantly; the dose ratio was 1.24. 4. None of the local infusions and/or systemic treatments had any significant effects on supine systolic and diastolic blood pressure and pulse rate. 5. DMI caused a substantial (47.6%) reduction in salivary output that significantly differed from the slight statistically insignificant increase (5.8%) of salivary output recorded after placebo. 6. These results show that a single oral dose (100 mg) of DMI causes significant potentiation of the response to noradrenaline without significantly affecting the response to methoxamine. The potentiation is likely to be due to uptake blockade since the response to methoxamine was not affected. Furthermore, the lack of significant antagonism of the response to methoxamine indicates that a single oral dose (100 mg) of DMI does not cause sufficient alpha 1-adrenoceptor blockade to be detected as a pharmacodynamic change in our test system.     

Vasoconstrictor responses to the P2x-purinoceptor agonist beta, gamma-methylene-L-ATP in human cutaneous and renal blood vessels.

1. Strips of human saphenous veins and of human renal arteries and veins were superfused with Krebs-Henseleit solution at 37 degrees C. Constrictor responses were elicited by exogenous noradrenaline and the P2x-purinoceptor-selective agonist, beta, gamma-methylene-L-ATP. 2. In human saphenous veins, beta, gamma-methylene-L-ATP (0.3-30 microM; EC50 2.2 microM) induced marked constrictor responses. The maximal response to beta, gamma-methylene-L-ATP was similar to the maximal response to noradrenaline. The P2-purinoceptor antagonist suramin (30 microM) shifted the concentration-response curve of beta, gamma-methylene-L-ATP to the right (apparent pKB value 4.8); suramin (100 microM) markedly inhibited the responses to beta, gamma-methylene-L-ATP. The preferential P2x-purinoceptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 3 microM) slightly reduced the response to beta, gamma-methylene-L-ATP. At a ten times higher concentration (30 microM), PPADS almost abolished the responses to beta, gamma-methylene-L-ATP. PPADS (30 microM), in contrast, caused no significant change in the concentration-response curve of noradrenaline. 3. In extrarenal and intrarenal arteries, EC50 values and maximal responses to noradrenaline were similar when compared with responses to noradrenaline in saphenous veins. Noradrenaline also constricted extrarenal veins. However, in contrast to the results obtained on saphenous veins, beta, gamma-methylene-L-ATP caused almost no constrictor responses in extrarenal veins and arteries and only moderate responses in intrarenal arteries. 4. The results demonstrate marked differences in responsiveness of human blood vessels to the selective P2x-purinoceptor agonist, beta, gamma-methylene-L-ATP, suggesting tissue differences in the occurrence or operation of P2x-purinoceptors in human vascular tissues. Moreover, the results indicate that PPADS blocks P2x-purinoceptors in human isolated blood vessels as previously demonstrated in animal blood vessels.     

The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization.

1. Organ bath experiments and measurements of prostanoids were performed to investigate the presence of nitric oxide synthase in venous smooth muscle and its interaction with cyclo-oxygenase. 2. In rings of canine saphenous vein without endothelium, the inhibitor of cyclo-oxygenase, indomethacin (10 microM), induced contraction. NG-nitro-L-arginine (100 microM) (L-NOARG), an inhibitor of nitric oxide synthase did not affect the tone of rings of canine saphenous vein when administered alone. However, in the presence of indomethacin L-NOARG (100 microM) induced further contraction. 3. Similar results were obtained in response to NG-monomethyl-L-arginine (L-NMMA)(300 microM or NG-nitro-L-arginine methylester (L-NAME)(100 microM). 4. When rings of canine saphenous vein without endothelium were contracted with phenylephrine (1 microM) instead of indomethacin, neither L-NOARG or L-NMMA induced further contraction. 5. When rings of canine saphenous vein without endothelium were contracted with noradrenaline (0.3 microM) in the presence of indomethacin (10 microM) plus L-NOARG (100 microM), a relaxation to L-arginine was observed. Transient relaxations to superoxide dismutase (150 u ml-1) were observed in all rings. 6. When rings of saphenous vein without endothelium were incubated with lipopolysaccharide (LPS) (100 micrograms ml-1) or interleukin-1 beta (10 u ml-1) the concentration-contraction curve to noradrenaline was not affected. 7. Rings without endothelium released prostaglandin E2 and prostaglandin I2, as measured by radioimmunoassay. The basal production was abolished by indomethacin and not affected by L-NOARG. 8. These results suggest that when cyclo-oxygenase is inhibited, a nitric oxide synthase activity is revealed in rings of canine saphenous vein without endothelium.     

Cutaneous venous dysfunction studied in vivo in the LPS-treated rabbit: implication of NO in saphenous vein hyporeactivity

Superficial vein pathology involves both mechanical (hyperpressure and distension) and inflammatory mechanisms. Conflicting results exist about the role of NO in the venous hyporeactivity induced by inflammation. In order to clarify this point, we aimed to investigate the effects of sepsis on cutaneous vein responsiveness in vivo and the possible contributions of constitutive and inducible NOS to the changes of venous contractility. Saphenous vein diameter was recorded by an ultrasonic echo-tracking device in pentobarbital-anaesthetised rabbits. Bacterial lipopolysaccharide (LPS) was administered i.v. at 20 mg/kg/15 min, inducing a progressive fall in mean arterial blood pressure after 2-3 h. The effects of LPS on saphenous vein responsiveness to noradrenaline (2 microg/kg i.v.) were measured simultaneously. In some rabbits, veins were removed for immunochemistry to detect iNOS staining. The venoconstriction to noradrenaline was already significantly reduced at 30 min after LPS (6+/-1% instead of 19+/-1% before LPS) and was completely abolished 3 h after LPS. A reduction of the venoconstriction induced by sumatriptan, a 5-HT(1B/D) agonist, (100 microg/kg, 11+/-1% after saline n=5) was also observed 180 min after LPS infusion (3+/-1%, n=4). The venodilatations induced by acetylcholine or sodium nitroprusside injected locally into the vein were not altered by LPS. When administered 90 min after LPS infusion, the NOS inhibitor L-NAME but not the selective iNOS inhibitor L-NIL (10 mg/kg) induced a recovery of the venoconstriction. Preventive perfusion with L-NAME (10 mg/kg/2 h) reduced the initial hyporeactivity to noradrenaline (30 to 60 min), but accelerated the lethal fall in MAP. L-NIL (10 mg/kg/2 h), to a lesser extent than L-NAME, also reduced the initial hyporeactivity to noradrenaline; in contrast to L-NAME, L-NIL also delayed the complete loss of noradrenaline constriction and improved animal survival. In control animals, neither L-NAME nor L-NIL modified the venoconstriction induced by noradrenaline. iNOS staining was observed in the saphenous vein endothelium after LPS. The experimental model developed in these experiments allows the study of venous responsiveness during sepsis in vivo. Our results show that LPS administration reduces saphenous vein contractility to both adrenergic and serotoninergic constrictor agents. The data suggest that both endothelial and inducible NO are involved in the loss of venous reactivity but these enzymes exert contrasting effects on blood pressure changes.     

The termination of action of catecholamines in the isolated venous tissue of the dog

Summary The termination of responses to noradrenaline and adrenaline was studied, using the oil immersion technique, in helically cut strips obtained from the lateral saphenous vein of the dog. Inhibitors of catechol-O-methyltransferase (tropolone), monoamine oxidase (iproniazid) or neuronal uptake (cocaine and imipramine-N-oxide) slowed the relaxation rate of strips contracted by noradrenaline or adrenaline (10^–5 and 10^–6 M) and immersed in oil. From these experiments it is concluded that, at the concentrations used, oxidative deamination represents the major inactivation pathway for both catecholamines, and that O-methylation plays only a minor role in the disposition of the amines; however, the inactivation of adrenaline is more dependent on catechol-O-methyltransferase than that of noradrenaline. Tissue uptake and storage occupied an intermediate position between the enzymatic pathways referred to: iproniazid inhibited 79%, cocaine 54% and tropolone 27% of the inactivation capacity when 10^–5 M noradrenaline was used. However, the combination of the three drugs inhibited only 88% of the inactivation capacity. This is due to the marked overlap of the effects of cocaine and iproniazid, which is attributed to the blockade by cocaine of the access of the amine to intraneuronal sites of oxidative deamination.Cocaine caused marked potentiation of responses to noradrenaline and moderate prolongation of relaxation time, while iproniazid, which had much more marked effects on the termination of action of noradrenaline, induced only a slight augmentation of the contractile responses. It is concluded that potentiation is an unreliable index of altered disposition of noradrenaline in this preparation.Experiments with perfused venous segments showed that, following the concentration gradient, noradrenaline diffuses freely through the vein wall, and that there is no marked difference in responses to adventitial or intimal application of the amine. Radioautographic studies gave evidence of two different types of accumulation (neuronal and extraneuronal), which appeared to be in equilibrium.Supported in part by a grant from III Plano de Fomento (Actividades, 1970).     

A comparison of the effects of angiotensin II and Bay K 8644 on responses to noradrenaline mediated via postjunctional alpha 1-and alpha 2-adrenoceptors in rabbit isolated blood vessels.

1. The effects of angiotensin II (AII) and Bay K 8644 on responses to noradrenaline (NA) mediated via postjunctional alpha 1- and/or alpha 2-adrenoceptors have been compared in three isolated venous preparations from the rabbit, the lateral saphenous vein, the left renal vein and the ear vein. 2. A similar action of AII and Bay K 8644 was observed only in the lateral saphenous vein; each potentiated responses to NA after isolation of a homogeneous population of postjunctional alpha 2- adrenoceptors. However, even in this preparation the mechanism of action for these agents was not identical. The sensitivity of KCl-induced contraction to changes in extracellular calcium ions (reflecting activation of voltage-dependent Ca2+ channels) was enhanced by Bay K 8644 but reduced by AII. 3. All produced a selective facilitation of responses mediated via postjunctional alpha 2-adrenoceptors. In the lateral saphenous vein it reduced the effectiveness of prazosin and facilitated responses after isolation of alpha 2-adrenoceptors with phenoxybenzamine and rauwolscine. It directly enhanced responses to NA in the ear vein, where only alpha 2-adrenoceptors are involved. In contrast, AII did not influence responses mediated via postjunctional alpha 1-adrenoceptors in the left renal vein (even after the receptor reserve had been removed with phenoxybenzamine) nor the 'rauwolscine-resistant' component of responses to NA in the saphenous vein. 4. Bay K 8644 enhanced contractile responses to NA mediated both via alpha 2-adrenoceptors, in the lateral saphenous vein, and via alpha 1-adrenoceptors in the left renal vein. Thus, unlike angiotensin II, no preferential effect was apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of desipramine on the uptake of mescaline by cortical synaptosomes

The uptake of ¹⁴C-labelled noradrenaline and mescaline into a synaptosome-rich preparation from the rat cerebral cortex was studied. Noradrenaline was accumulated by the synaptosome-rich preparation by a temperature-dependent process which was competitively inhibited by desipramine. Mescaline was accumulated by a temperature- and sodium-dependent process. The uptake of mescaline was not affected by desipramine. The uptake of noradrenaline was inhibited by mescaline in a non-competitive manner; this suggests that the uptake of mescaline is not brought about by the noradrenaline uptake mechanism.     

The potassium channel opening drug cromakalim produces arterioselective vasodilation in the upper limbs of healthy volunteers

1. The effect of the K+ channel opening drug cromakalim on forearm blood flow during direct infusion into the brachial artery, and on the size of noradrenaline preconstricted hand veins during infusion directly into the vein, was studied in eight healthy volunteers. 2. Cromakalim (0.01-10.0 micrograms min-1) produced a dose-dependent increase in blood flow in the infused forearm, from 5.4 +/- 2.5 to 15.1 +/- 7.3 ml 100 ml-1 forearm min-1 at 10 micrograms min-1 (P less than 0.001). The half-time of offset of its action was 30 min. There was no change in blood flow in the non-infused forearm. 3. There was no increase in the size of noradrenaline pre-constricted dorsal veins during local infusion of cromakalim (0.001-1.0 microgram min-1). Glyceryl trinitrate (0.4 microgram min-1) however, completely reversed the constriction to noradrenaline (P less than 0.001). 4. The clear arterioselectivity of cromakalim, as with other members of this new class of drug, accords with the dependency of venoconstriction on receptor-operated, rather than potential-operated mechanisms which are of importance in resistance vessels. With this haemodynamic profile cromakalim may prove of value in the treatment of arterial hypertension.     

Effect of lofepramine and other antidepressants on the uptake of 5-hydroxytryptamine and noradrenaline into rat brain monoaminergic neurons.

Lofepramine, (N-methyl-N-[4-chlorobenzoylmethyl]-3-[10,11-dihydro-5H-dibenz(b,f)-azepin-5-yl]-propylamine hydrochloride), is a new antidepressant with low toxicity and no peripheral anticholinergic activity. Its effect on 5-hydroxytryptamine (5-HT) and noradrenaline uptake into rat brain monoaminergic neurons was studied and compared with that of other antidepressants, particularly with that of imipramine and desipramine. Lofepramine inhibited both 5-HT and noradrenaline uptake into synaptosomal fractions in vitro but was 4 times more potent in inhibiting noradrenaline than 5-HT uptake, indicating the effect resembles that of desipramine. Noradrenaline uptake was also preferentially inhibited in synaptosomes from brain of rats treated previously with lofepramine or desipramine (i.p.). Pretreatment with SKF 525A (i.p.) did not diminish the effect of lofepramine, but rather potentiated it. Therefore it is suggested that the formation of desipramine is not necessary for lofepramine to exhibit, the effect on amine uptake in vivo. Both lofepramine and desipramine inhibited intraventricular noradrenaline uptake into synaptosomes without any effect on 5-HT uptake. These results suggest that lofepramine is qualitatively similar to desipramine with respect to preferential inhibition of noradrenaline uptake into central noradrenergic neurons.     

Arrhythmias and inhibition of noradrenaline uptake caused by tricyclic antidepressants and chlorpromazine on the isolated perfused rabbit heart

1. Isolated rabbit hearts were perfused with a modified Tyrode solution containing noradrenaline in concentrations increasing stepwise from 5.9 nM to 5.9 muM at 5 min intervals. This dose regime was applied twice before and once 20 min after starting perfusion with one of 9 tricyclic drugs. Ventricular rate and right atrial and ventricular tensions were recorded using the transverse method. 2. Infusions of noradrenaline evoked ventricular arrhythmias in hearts perfused with amitriptyline 4.8 muM, chlorpromazine 5.0 muM, desipramine 5.0 muM, dibenzepine 34.7 muM, doxepin 4.7 muM, imipramine 4.7 muM, noxiptiline 9.1 muM and opipramole 9.2 muM. The incidence of arrhythmias increased with the concentration of noradrenaline applied and the dose of tricyclic drug administered. Whenever arrhythmias had started they continued as long as noradrenaline was infused. Noradrenaline failed to produce arrhythmias in hearts not exposed to drugs and after iprindole 4.7 muM or cocaine 2.9-18 muM. 3. Propranolol 0.1 muM inhibited the incidence of arrhythmias after doxepin 4.7 muM plus noradrenaline 5.9-190 nM. 4. Neuronal uptake of exogenous noradrenaline in the rabbit heart was inhibited by the tricyclic drugs in the following order of declining p potency: doxepin, noxiptiline, amitriptyline, desipramine, chlorpromazine, imipramine, dibenzepine, opipramole and iprindole. 5. Among tricyclic drugs the potency to inhibit amine uptake is related to the incidence of arrhythmias evoked by a submaximal concentration of noradrenaline. It appears, however, that these two parameters are not causally linked. 6. The isolated rabbit heart perfused with noradrenaline might be used as a model for testing the arrhythmogenic actions of tricyclic drugs and the treatment of such arrhythmias.