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Females have increase in-hospital mortality and poorer outcomes following coronary artery bypass grafting (CABG). Biological differences in the reactivity of the graft conduits to circulating catecholamine may contribute to this sex difference. This study examined sex differences in the vasoconstrictor responses of internal mammary artery (IMA) and saphenous vein (SV) conduits to phenylephrine (PE) and endothelin-1 (ET-1). Functional IMA and SV were obtained from 78 male and 50 female patients undergoing CABG (67.7 ± 11 and 69 ± 10 years, respectively) and subjected to the following experimental conditions. (1) Concentration response curves for PE and ET-1 were generated in an intact IMA and SV and endothelium denuded IMA segments, (2) in the presence of the nitric oxide synthase inhibitor (L-NAME) or the cyclooxygenase inhibitor (indomethacin) in an endothelium-intact IMA and (3) the activity state (abundance and phosphorylation) of the α 1 -adrenergic receptor was investigated using Phos-tag™ western blot analysis. (1) Compared to male, female IMA and SV were hypersensitive to PE but not ET-1 (p < 0.05). The female IMA hypersensitivity response to PE was abolished following endothelial denudation, (2) persisted in the presence of L-NAME but was abolished in the presence of indomethacin and (3) there was no sex differences in the abundance and phosphorylation of the α 1 -adrenergic receptor in IMA. Female IMA and SV graft conduits are hypersensitive to α 1 -adrenergic stimuli. This endothelial cyclooxygenase pathway-mediated hypersensitivity may produce excessive IMA and SV graft constriction in females administered catecholamines and could contribute to their poorer CABG outcomes.

Recent findings have suggested that the primary factors for development of chronic venous disease (CVD), which commonly manifests as varicose veins (VV), are due to structural and biochemical modifications of the vessel wall. The aim of this exploratory study was to characterize by flow cytometry the endothelial cells (EC) mechanically extracted from the varicose saphenous veins (VSV) segments of patients submitted to VV surgery, and to compare the expression of cell surface molecules in these EC with that observed in the EC from the graft SV (GSV) of patients undergoing bypass surgery. EC were isolated from distal- (varicose trunk) and from proximal- (nearly normal) VSV segments of 30 patients submitted to VV surgery, and from proximal GSV segments of 20 patients submitted to bypass surgery (control group), using a mechanical method, and their immunophenotype was characterized by flow cytometry. EC were identified as being CD45 neg CD146 bright CD31 bright , and analyzed for expression of activation-related (CD54, CD62E, CD106), procoagulant (CD142), and cell junction (CD31, CD146) molecules, and for the scavenger receptor, CD36. The EC harvested from the SV segments of CVD patients had lower expression of all the molecules evaluated, in comparison to controls; these differences were more evident for the EC isolated from the distal-VSV. The EC extracted from the proximal- and distal-VSV segments of the CVD patients also differ from each other, the first having lower levels of CD62E, CD106, CD142 and CD36. Groups did not match for gender and controls were heterogeneous concerning the underlying pathologies, which may have a confounding effect. Our study revealed that the EC isolated from varicose (distal) and nearly normal (proximal) VSV segments of the CVD patients differ phenotypically from each other, and from the EC of the control group. The VSV segments more affected by the CVD have the lowest expression of the studied markers. We hypothesize that CVD is associated with a decrease on the EC surface molecules, causing EC dysfunctionality. Further studies with a large number of gender-matched participants are needed, to confirm the results obtained in this exploratory study.

Aims/hypothesis: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide., Methods: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance., Results: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days)., Conclusions/interpretation: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.

Vasoconstriction to agonists at serotonin (5-hydroxytryptamine; 5-HT) receptors and alpha-adrenoceptors, as well as vasodilatation induced by alpha-CGRP, have been well described in the porcine carotid circulation in vivo. The present study sets out to investigate the effects of current and prospective antimigraine drugs on porcine meningeal artery segments in vitro. Sumatriptan, ergotamine, dihydroergotamine, isometheptene and clonidine failed to contract the meningeal artery, but 5-HT, noradrenaline and phenylephrine induced concentration-dependent contractions. The contractions to 5-HT were competitively antagonized by the 5-HT(2A) receptor antagonist ketanserin, whilst those to noradrenaline were antagonized by alpha(1)-(prazosin), alpha(2)-(rauwolscine and yohimbine) and alpha(2C/2B)-(OPC-28326) adrenoceptor antagonists. Whilst dobutamine and salbutamol were ineffective, alpha-CGRP produced concentration-dependent relaxations that were antagonized by the CGRP(1) receptor antagonist olcegepant. In agreement with their lack of contractile effect, sumatriptan and ergotamine failed to influence forskolin-stimulated cyclic AMP accumulation in the porcine meningeal artery; in contrast, both compounds decreased forskolin-stimulated cyclic AMP accumulation in the human isolated saphenous vein, where they induced contractions. Finally, using RT-PCR, we could demonstrate the presence of mRNAs encoding for several 5-HT receptors (5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(2A) and 5-HT(7)) and adrenoceptors (alpha(1A), alpha(1B), alpha(1D), alpha(2A), alpha(2B), alpha(2C), beta(1) and beta(2)), as well as that for the calcitonin receptor like receptor, a component of the CGRP(1) receptor. These results suggest that: (i) the porcine meningeal artery may not be involved in the vasoconstriction of the carotid vascular bed elicited by antimigraine drugs in anaesthetized pigs, and (ii) the mismatch between the presence of receptor mRNA and the lack of response to sumatriptan, dobutamine and salbutamol implies that mRNAs for the 5-HT(1B) receptor and beta(1)- and beta(2)-adrenoceptors are probably unstable, or that their density is too low for being translated as receptor protein in sufficient quantities.

Although saphenous veins and internal mammary arteries are commonly used for coronary artery bypass grafting, only a very few comparative studies are available on alpha-adrenoceptor-mediated vasoconstriction in these vessels. Thus, we determined, in isolated rings from human saphenous vein and human internal mammary artery, contractile responses to noradrenaline (10(-8)-10(-4) M) in the absence and presence of the alpha-adrenoceptor antagonists yohimbine (alpha(2)-adrenoceptor antagonist, 10(-8)-10(-6) M), prazosin (alpha(1)-adrenoceptor antagonist, 10(-9)-10(-7) M), 5-methyl-urapidil (5-MU, alpha(1A)-adrenoceptor antagonist, 10(-8)-10(-6) M), BMY 7378 (alpha(1D)-adrenoceptor antagonist, 10(-7)-10(-6) M), and chloroethylclonidine (CEC, irreversible alpha(1B)-adrenoceptor antagonist, 3x10(-5) M for 30 min). All experiments were carried out in the presence of 10(-7) M propranolol and 10(-5) M cocaine. In both vessel types noradrenaline evoked concentration-dependent contractions. In saphenous veins yohimbine was a potent antagonist (pA(2)-value 8.32) while prazosin, 5-MU and BMY exhibited only marginal antagonistic effects. CEC, however, significantly decreased noradrenaline-induced contractions. In contrast, in internal mammary arteries prazosin (pA(2)-value 9.65) and 5-MU (pK(B)-values 7.2-7.5) were potent antagonists, while yohimbine and BMY exhibited only weak antagonistic effects. CEC, however, significantly decreased noradrenaline-induced contractions. We conclude that in saphenous vein the contractile response to noradrenaline is mediated predominantly by alpha(2)-adrenoceptors, while in internal mammary artery it is mediated (to a major part) by alpha(1B)- and (to a minor part) by alpha(1A)-adrenoceptors.

The aim of the study was to determine the subtype of prejunctional alpha2-autoreceptors in the canine saphenous vein. Segments of the vein were incubated with 3H-noradrenaline and subsequently perifused with modified Krebs-Henseleit solution. Five periods of electrical stimulation were applied (S1-S5; each for 2 min, 1 Hz). Concentration-response curves for the inhibitory effect of the alpha2-adrenoceptor agonists oxymetazoline and UK-14,304 and for the facilitatory effect of nine antagonists were determined. Correlations between the pEC30%s for the antagonists obtained in the present study and the pKis for the same antagonists obtained in tissues expressing only alpha2A- (HT29 cells), alpha2B- (neonatal rat lung), alpha2C- (OK cells) or alpha2D-adrenoceptors (rat submaxillary gland) showed that the alpha2-autoreceptors in the canine saphenous vein resemble most closely the alpha2A-subtype. Furthermore, oxymetazoline was a highly potent agonist (pIC50% = 8.10) and prazosin was a weak antagonist (pEC30% = 6.46), confirming that the alpha2-adrenoceptors involved in the modulation of the response to electrical stimulation of the canine saphenous vein do not belong to either the alpha2B- or alpha2C-subtype. On the other hand, the EC30% ratios phentolamine/rauwolscine and idazoxan/rauwolscine were much closer to the ratios obtained at alpha2-autoreceptors of the rabbit- (alpha2A) than of the guinea-pig brain cortex (alpha2D). The results suggest that the sympathetic nerves of the canine saphenous vein are endowed with alpha2A-adrenoceptors.

The pharmacological properties of the presynaptic a2-autoreceptors mediating inhibition of noradrenaline release were investigated in human saphenous vein and rabbit pulmonary artery. Segments of these blood vessels were incubated with [3H]noradrenaline and subsequently superfused with physiological salt solution containing uptake1 and uptake2 blockers. The potencies of a2-adrenoceptor antagonists in facilitating (pEC40) the electrically (2 Hz) evoked tritium overflow were determined. The order of potency and potency ratios of a2-adrenoceptor antagonists obtained in our experiments were compared with the corresponding order of affinity and affinity ratios from radioligand binding studies in tissues and cells expressing only one of the alpha 2-adrenoceptor subtypes. In the rabbit pulmonary artery, oxymetazoline was a highly potent agonist at presynaptic a2-adrenoceptors, as reflected by its ability to inhibit at low concentrations the electrically evoked tritium overflow. However, in the human saphenous vein oxymetazoline behaved as a partial agonist, which, in interaction experiments with the a2-adrenoceptor agonist B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine), exhibited high potency in antagonizing the inhibitory effect of the latter drug on tritium overflow. Prazosin given alone at concentrations up to 1 mumol/l did not affect tritium overflow. The data obtained with oxymetazoline and prazosin make it very improbable that the a2-autoreceptors on the sympathetic nerves in both tissues are of the a2B- or a2C-subtype. In both blood vessels, rauwolscine given alone was highly potent in facilitating the electrically evoked overflow. In agreement with this, rauwolscine exhibited high potency in antagonizing the inhibitory effect of oxymetazoline on tritium overflow in the rabbit pulmonary artery and of B-HT 920 in the human saphenous vein. The ratio phentolamine/rauwolscine calculated from their potencies in increasing the electrically evoked tritium overflow was also used to discriminate between the various a2-adrenoceptor subtypes. Comparison of this potency ratio with the corresponding affinity ratios for a2-adrenergic binding sites on HT 29 cells, human platelets, bovine pineal gland, rat submaxillary gland, and cell lines transfected with the human a2 genes indicates that in the rabbit pulmonary artery and human saphenous vein the pharmacological characteristics of the autoreceptors conform best to those of a2A-adrenoceptors. Finally, in both blood vessels the potencies of the antagonists BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), rauwolscine, corynanthine, phentolamine, idazoxan, SKF 104078 (6-chloro-9-[(3-methyl-2-butenyl) oxyl]-3-methyl-1H-2,3,4,5-tetrahydro-3-benzazepine), and/or tolazoline in facilitating evoked noradrenaline release was determined. The potencies of these drugs which can discriminate between a2A- and a2D-adrenoceptors (but not between these and a2B/2C-adrenoceptors) were correlated significantly with their affinities for a2A, but not a2D, sites in radioligand binding studies. In conclusion, the present results suggest that the sympathetic nerves of the human saphenous vein and rabbit pulmonary artery are endowed with a2-autoreceptors of the a2A subtype.

The neurotransmitter, noradrenaline, is inactivated by active transport out of the synaptic cleft, either back into the adrenergic neurons (uptake1) or into extraneuronal cells (uptake2). Although experimental models clearly demonstrate uptake2 in various animal tissues, there are few reports of studies that demonstrate uptake2 in human tissue. Furthermore, uptake2 has never been described in human heart tissue. We demonstrated a reduction of 3H-noradrenaline uptake in human atrial and venous tissue and in rat atrial and ventricular tissue by O-methyl isoprenaline-a known inhibitor of uptake2. These results indicate that uptake2, as has been described in previous experimental models, does exist in humans and further, the results emphasize the importance of uptake2, since about 50% of exogenous noradrenaline is inactivated by uptake2 in the rat heart.

This study was aimed at analysing the contractile response of the dog saphenous vein to chloroethylclonidine. At 37 degrees C, chloroethylclonidine (0.1-100 mumol.l-1) caused a long-lasting contraction in both proximal and distal segments of the dog saphenous vein, reaching 77.6 and 52.6% of the maximal response to phenylephrine, respectively. At 18 degrees C, and in both segments, the maximal response to chloroethylclonidine was markedly reduced, whereas that to phenylephrine was not changed and that to UK-14,304 was enhanced. The response to chloroethylclonidine was unaffected by pretreatment with cocaine. Warming to 37 degrees C caused contraction of strips which at 18 degrees C had remained unresponsive to chloroethylclonidine, even if these strips were repeatedly washed before warming. At 18 degrees C, chloroethylclonidine (100 mumol.l-1) did not alter the responses to UK-14,304 and phenylephrine. At 37 degrees C, the contractile response to chloroethylclonidine was antagonized by yohimbine, rauwolscine and prazosin, with the potency rank yohimbine = rauwolscine > prazosin. Phenoxybenzamine (30 nmol.l-1) displaced the concentration-response curve to chloroethylclonidine to the right and depressed its maximum. After phenoxybenzamine, yohimbine continued to be more effective than prazosin, which remained very potent.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of 3H-noradrenaline and formation of 3H-metabolites were studied in the saphenous vein of newborn (mean age, 18 h) and adult dogs. Vein strips were incubated with 0.23 mumol/l of 3H-noradrenaline during 1 h and washed out for 110 min; thereafter, the perifusion fluid was collected in 5-min samples. Electrical stimulation was applied at 120 min (1 Hz, 2 ms, 100 V, for 5 min). In some experiments the tissues were preincubated with 1 mmol/l pargyline (to inhibit monoamine oxidase). In these experiments, 12 mumol/l cocaine (to inhibit uptake1), 41 mumol/l hydrocortisone (to reduce uptake2) and 50 mumol/l U-0521 (to inhibit COMT) were present during the perifusion. 3H-noradrenaline, 3H-DOPEG, 3H-NMN, 3H-DOMA and 3H-OMDA were separated by column chromatography. The noradrenaline content of the tissue was estimated by HPLC followed by electrochemical detection. A morphological study was also carried out by light and electron microscopy. The endogenous noradrenaline content of the saphenous vein was 4.3 times higher in adults than in neonates. The number of varicosities was similar in adults and newborns but the number of vesicles per varicosity profile was 5 times higher in adults. Hence, the endogenous noradrenaline content per vesicle was about the same in adults and newborns. The accumulation of 3H-noradrenaline per vesicle was about 5 times higher in newborns than in adults. On the other hand, the vein wall media of neonates was about 3 times thinner than that of adults. The evoked fractional release of tritium was about 10 times higher in neonates than in adults, whether the inactivation pathways were blocked or not.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of N- and L-type voltage-dependent Ca channels (VDCCs) in adrenergic neurotransmission under the superfusion with 0.25 mM Ca2+ + 20 mM tetraethylammonium (low Ca2+ + TEA) medium has been studied by examining the effects of omega-conotoxin GVIA (omega-CTX) and dihydropyridine antagonists and agonist on transmural nerve stimulation (TNS)-evoked 3H overflow from canine saphenous veins preloaded with [3H]-noradrenaline. Nisoldipine (10 and 30 microM) and nifedipine (30 microM) reduced significantly the TNS-evoked 3H overflow in low Ca2+ + TEA medium, while the two dihydropyridine antagonists failed to suppress it in normal Krebs medium. Bay K 8644 (30 and 100 nM) produced a significant and concentration-dependent enhancement of the TNS-evoked 3H overflow in low Ca2+ + TEA medium. The enhancing effects of Bay K 8644 were antagonized by both 3 microM nisoldipine and 10 microM nifedipine. omega-CTX inhibited markedly the TNS-evoked 3H overflow in both normal Krebs and low Ca2+ + TEA media, the inhibition by omega-CTX being ten times more potent in low Ca2+ + TEA medium. Nisoldipine (30 microM), when combined with 1 nM omega-CTX, produced a further significant inhibition of the TNS-evoked 3H overflow in low Ca2+ + TEA medium. However, no additional inhibition by 30 microM nisoldipine was observed when omega-CTX concentration was raised to 2 nM. In the veins superfused with normal Krebs medium, nisoldipine (30 microM) did not affect the inhibitory effect of 10 nM omega-CTX on the evoked 3H overflow.(ABSTRACT TRUNCATED AT 250 WORDS)

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.(ABSTRACT TRUNCATED AT 250 WORDS)

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.(ABSTRACT TRUNCATED AT 250 WORDS)

The augmentation by the cardioactive steroid acetylstrophanthidin of neurotransmitter release evoked by tyramine, and the dependence of the augmentation upon Na+, has been investigated in dog saphenous vein rings in which monoamine oxidase activity and uptake2 had been inhibited with pargyline and corticosterone respectively. High extracellular Na+ (Nao; 263 mmol/l) reduced basal efflux of 3H-compounds from the rings and also reduced tyramine-evoked efflux. Low Nao (25 mmol/l) increased basal efflux of 3H but reduced tyramine-evoked efflux. The increment in basal 3H-efflux caused by low Nao was cocaine-sensitive. A presumed increase in intracellular Na+ (Nai), produced by preincubating rings with acetylstrophanthidin in normal (143 mmol/l) or high Nao, augmented 3H-efflux evoked by subsequent incubation with tyramine in normal Nao. Pre-incubating rings with acetylstrophanthidin in low Nao, conditions which would not be expected to increase Nai, did not cause augmentation of the subsequent tyramine-evoked 3H-efflux. An increase in Nai, produced either as above or by pre-incubating rings in high Nao alone, reduced subsequent neuronal 14C-tyramine uptake. Low Nao present only during incubation reduced neuronal 14C-tyramine uptake, but high Nao present only during incubation did not increase neuronal 14C-tyramine uptake from that measured in normal Nao. The data are consistent with the following hypotheses: that tyramine uptake is dependent upon the prevailing inwardly directed Na+-gradient, that consequent noradrenaline efflux is Na+-gradient dependent and that the enhancement by acetylstrophanthidin of tyramine-evoked 3H-efflux is a consequence of the raised Nai caused by Na+,K+-ATPase inhibition.

Dog saphenous vein strips were incubated with 2 muM 3H-isoprenaline. After 2 and 8 min of incubation small cross sections were cut and prepared for light and ultrastructural autoradiography. In addition, the accumulation of 3H-isoprenaline (3H-ISO) in the tissue as well as the formation of 3-O-methylisoprenaline (3H-OMI) were determined by liquid scintillation counting. The density of silver grains was 6.5 times higher on smooth muscle than on connective tissue. It decreased from the intima to adventitia when determined in the 2 min specimens, suggesting the existence of a selective transport of isoprenaline through the endothelium of the vein. The preferential distribution into the juxta-intimal part of the media which was observed after the 2 min incubation period was abolished by cortexone. Silver grain density, especially over the smooth muscle, was markedly increased by U-0521 and decreased by cortexone. Simultaneous treatment with cortexone and U-0521 led to a marked accumulation of silver grains over neuronal tissue; this accumulation was prevented by cocaine. Strips incubated at 0 degrees C and without oxygen showed almost no grains. In control conditions the saphenous vein strips removed 1968 +/- 19 pmoles-g-1 during 8 min of incubation. 86.3% of that value corresponded to 3H-OMI formation. O-methylation of 3H-ISO was strongly inhibited by U-0521 and, in a lesser degree, by cortexone. The influence of U-0521 on inhibition of O-methylation of 3H-ISO was not modified by cortexone. We found a linear correlation between silver grain densities per unit area of the vein strips and the 3H-isoprenaline accumulation determined by liquid scintillation counting, indicating a clear correspondence between silver grains and 3H-isoprenaline.

Specific inhibitors of MAO A (clorgyline 0.1 mumol/l) and of MAO B [(-)deprenyl 10 mumol/l] were used in dog saphenous vein strips in order to study the relative influence of the two types of MAO on: 1. Termination of contractile response to exogenous noradrenaline (NA); 2. Metabolism and accumulation of exogenous (3H)-NA; 3. Metabolism of 3H-NA released by electrical stimulation. To study the termination of contractile response to exogenous NA, the oil immersion technique was used to determine the time for half-relaxation. The experiments were performed on strips with or without treatment with cocaine 10 mumol/l (to inhibit neuronal uptake) plus U-0521 100 mumol/l (to inhibit catechol-O-methyl transferase) before and after exposure to MAO inhibitors. Clorgyline, but not (-)deprenyl enhanced significantly the time for half-relaxation of the strips, whether cocaine was present or not. To study the metabolism and accumulation of exogenous 3H-NA, the strips were incubated (with or without preincubation with cocaine) with 3H-NA 0.23 mumol/l and 2.3 mumol/l in the presence and in the absence of MAO inhibitors. The formation of deaminated metabolites was significantly reduced by clorgyline, but not by deprenyl. To study the metabolism of 3H-NA released by electrical stimulation, the strips were incubated with 3H-NA 1.4 mumol/l. In the presence of cocaine and U-0521, field stimulation was applied during two periods of 5 min (10 Hz, 100 V, 2 ms), in the absence or presence of MAO inhibitors. Under these experimental conditions clorgyline, but not deprenyl, abolished DOPEG formation, without affecting the other metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

The uptake and metabolism of 3H-5-hydroxytryptamine (5-HT) in the isolated saphenous vein of the dog was studied, as well as the sensitivity to 5-HT and its termination of action (as determined by oil immersion experiments). After exposure of the vein to 0.23 mumol/1 5-HT for 30 min, about one half of the 5-HT removed was accumulated in the tissue, the other half appearing in the form of metabolites. Cocaine, chlorimipramine or denervation reduced the accumulation, but not the metabolism, of 5-HT; hydrocortisone reduced both accumulation and metabolism. Hydrocortisone plus cocaine (or phenoxybenzamine alone) caused a greater reduction of accumulation and metabolism than hydrocortisone or cocaine alone. Pargyline caused an increase in accumulation and virtually abolished metabolism; amezinium also abolished metabolism, but reduced accumulation (the same effects as caused by cocaine plus pargyline). Cocaine (12 mumol/l) caused supersensitivity to 5-HT and a prolongation of the half-time for relaxation in oil; hydrocortisone (40 mumol/l) had no effect on sensitivity, but prolonged somewhat the half-time for relaxation in oil, showing supra-additive effects with cocaine. 5-HT, taken up into adrenergic nerve terminals, was released by electrical stimulation; no appreciable reduction in fractional release occurred during repeated stimulations. Exposure to cocaine prior to incubation with 5-HT prevented the neuronal uptake of 5-HT. Compartmental analysis, done with the help of washout curves of strips pretreated with pargyline and loaded with 3H-5-HT, shows that 3H-5-HT distributed into 3 compartments plus a bound fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (t50) (both in oil and in Krebs solution) of these agonists. Preliminary experiments have shown that the t50 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 muM) or in moderately high (2.3 muM) 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 in Krebs solution and by using cortexone (60 muM) 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.

Dog saphenous vein strips were incubated with 1.4 mumol/l 3H-(--)-noradrenaline for 60 min, after inhibition of the noradrenaline-metabolizing enzymes and of extra-neuronal uptake. At the end of the incubation period the strips were perifused for 150 min; cocaine (10 mumol/l) was added to the perifusion fluid from t = 75 min onwards. In some experiments either phentolamine (10 mumol/l) or clonidine (0.1 mumol/l) was also added at this time. Some strips were subjected to electrical stimulation from t = 100 to 150 min of perifusion (t = 0 being the start of perifusion), with frequencies ranging from 0.5 to 13.5 Hz. A compartmental analysis of spontaneous or electrically-induced efflux of 3H-noradrenaline was made. The spontaneous efflux had a long half time (t/2 = 124 min) and most of the 3H-noradrenaline which had accumulated in the strips did not participate in the efflux ("bound fraction", representing 90% of tissue activity at t = 100 min of perifusion). Neither phentolamine nor clonidine modified the half time or the "bound fraction" observed for spontaneous efflux. Electrical stimulation (greater than 0.5 Hz) mobilized only one compartment of noradrenaline, which represented about 50% of the noradrenaline accumulated in the strips. The half time of 3H-efflux induced by electrical stimulation decreased when the frequency increased from 0.5 Hz up to 13.5 Hz. Phentolamine increased the rate of efflux for all frequencies of stimulation and decreased the half time of efflux. However, the releasable pool of noradrenaline was only increased by phentolamine at 0.5 Hz, but not at higher frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments on the isolated saphenous vein of the rabbit have been performed in order to determine whether a coexistence of postjunctional alpha 1- and alpha 2-adrenoceptor subtypes can be demonstrated also under in vitro conditions. Prazosin, selective for alpha 1-receptors, and rauwolscine, selective for alpha 2-receptors, were used to antagonize the contractile response of the agonists phenylephrine (alpha 1), B-HT 920 (alpha 2) and noradrenaline (alpha 1/alpha 2). Each of the antagonists was equipotent against all three agonists; the effect of neither antagonist fulfilled the criteria for a competitive antagonism. The preferential alpha 1-receptor antagonist phenoxybenzamine, applied for irreversible blockade of alpha-receptors, reduced the effect of phenylephrine and B-HT 920 to the same degree. Thus, the results obtained with alpha-receptor antagonists cannot be reconciled with the coexistence of alpha 1- and alpha 2-receptors or the existence of only one of them. Contractile responses induced by alpha 1- and alpha 2-receptor agonists, respectively, could be differentiated by the calcium entry blocker nitrendipine. The effect of B-HT 920 was decreased whereas that of phenylephrine was hardly affected. Furthermore, we investigated the influence of angiotensin II on the effects of the alpha-adrenoceptor agonists as well as the antagonists. We observed firstly, that angiotensin, acting postsynaptically, potentiates the contractile response of certain alpha 2-receptor agonists and secondly, that in the presence of angiotensin the characteristics of the receptors as revealed by B-HT 920 are converted to typical alpha 2-adrenoceptors. It is concluded that the postjunctional alpha 2-receptors of the saphenous vein require the blood borne substance angiotensin for their expression.

Experiments were designed to study the release of newly synthesized catecholamines in the isolated canine saphenous vein. Unlabelled (--)-tyrosine did not affect the contractions caused by electrical stimulation or exogenous noradrenaline and did not influence the basal efflux and the stimulation-induced overflow of 3H-noradrenaline in veins, previously incubated with the radiolabelled transmitter. The precursor increased the stimulation-induced overflow of the principal intraneuronal metabolite of 3H-noradrenaline, 3H-3,4-dihydroxyphenylglycol, and augmented the concentration of endogenous dopamine. Available column chromatographic procedures were modified to measure small amounts of 3H-catecholamines in the presence of large concentrations of tritiated (--)-tyrosine. Incubation of isolated veins with 3H-(--)-tyrosine caused concentration- and time-dependent tissue accumulation of newly synthesized catecholamines; the amounts of 3H-noradrenaline and 3H-dopamine were roughly comparable and were augmented by raising the external K+ concentration. In isolated veins, first incubated with 3H-(--)-tyrosine, and then superfused, a small basal efflux of 3H-noradrenaline was detected. Electrical stimulation caused a frequency-dependent overflow of 3H-catecholamines consisting mainly of 3H-noradrenaline; a stimulation-evoked efflux of 3H-dopamine (10% of total 3H-catecholamines) could be detected only when long stimulation periods were applied. After a 70 min period of superfusion, the 3H-dopamine content of the tissues decreased while that of 3H-noradrenaline remained unchanged, irrespective of whether the tissues had been stimulated electrically or not. The present results show that the isolated canine saphenous vein can synthesize and release 3H-noradrenaline and 3H-dopamine when incubated with 3H-(--)-tyrosine.

Responses of canine saphenous veins to bradykinin and angiotensin and the effect of cyclosporine-A were investigated both in conscious dogs in vivo and on ring preparations from canine saphenous veins in vitro. In vivo local infusion of bradykinin into the saphenous vein elicited dose-dependent reduction in compliance, i.e., venoconstriction, whereas local infusion of angiotensin elicited dose-dependent venodilatation, which was markedly enhanced during blockade of endogenous thromboxane A2 synthesis by dazoxiben (2.5 mg/kg i.v.). The venoconstrictor response to bradykinin was attenuated after oral administration of both the thiazide-like diuretic clopamide (0.5 mg/kg) or cyclosporine-A (30 mg/kg), and by concomitant local infusion of cyclosporine-A (1-10 micrograms/min). Systemic i.v. infusion of the renin inhibitor H-77 (0.1 mg/kg/h) reversed the inhibition of bradykinin by both clopamide and cyclosporine-A. In vitro bradykinin elicited relaxation at low (0.1-10 nmol/l) but constriction at higher concentrations. The venoconstrictor response to bradykinin was resistant to blockade of thromboxane A2 synthesis and only partially attenuated after selective blockade of cyclooxygenase or lipoxygenase. Concomitant blockade of both lipoxygenase and cyclooxygenase activity by nordihydroguaiaretic acid (NDGA 10-30 mumol/l) nearly abolished the contractile response thereby enhancing the relaxant component of the bradykinin effect. Angiotensin II also elicited biphasic responses of partially contracted venous rings. Concomitant blockade of both lipoxygenase and cyclooxygenase by NDGA (10 mumol/l) again attenuated the contractile component of the angiotensin effect thereby unmasking the venodilator activity which could be inhibited by the angiotensin II receptor blocker saralasin (0.01-1 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

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 (O-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.

The affinities of 16 5-hydroxytryptamine (5-HT) receptor agonists (indole derivatives) and 7 5-HT receptor antagonists for [3H]5-hydroxytryptamine [( 3H]5-HT) binding sites in rat cerebral cortex membranes were determined. In addition, the potencies of the agonists for inhibiting the electrically induced tritium overflow from rat brain cortex slices preincubated with [3H]5-HT and from canine saphenous veins preincubated with [3H]noradrenaline were measured. Furthermore, the potencies of the indole derivatives for inducing contractile responses of canine saphenous veins were recorded. In addition, the interaction of the antagonists with unlabelled 5-HT at the 5-HT autoreceptor was studied in rat brain cortex slices. There was a good correlation between the binding affinities of the indole derivatives for the [3H]5-HT sites of rat brain cortex membranes and their potencies for inhibiting the evoked tritium overflow from both rat brain cortex slices and strips of canine saphenous vein. Comparison of the inhibition constants derived from the overflow experiments in both tissues again revealed a high correlation coefficient while there was only weak correlation between the binding affinities in rat brain cortex and the contractile potencies of the drugs in canine saphenous vein strips. When 5-HT receptor antagonists were investigated, metitepin and metergoline showed moderate affinities for the 5-HT autoreceptors in rat brain cortex slices, whereas quipazine had only weak affinity, and ketanserin, metoclopramide, cinanserin and cyproheptadine exhibited no antagonistic property. In binding experiments, the competition curves of most 5-HT receptor antagonists were biphasic, suggesting that the [3H]5-HT binding sites are heterogeneous.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments were designed to determine the effect of monoamine oxidase (MAO) inhibitors on the release and the metabolism of noradrenaline in the canine saphenous vein. Helical strips were incubated with 3H-noradrenaline and mounted for superfusion and measurement of the efflux of labelled transmitter and its metabolites; in certain experiments the tissue content of 3H-noradrenaline and its metabolites was also determined. The MAO-A inhibitor clorgyline, and the non-specific inhibitor pargyline, but not the MAO-B inhibitor deprenyl decreased the appearance of deaminated and O-methylated deaminated metabolites under basal conditions and during electrical stimulation. The MAO-A and the non-specific MAO inhibitor did not decrease the efflux of VMA to the same extent as that of the other deaminated metabolites. During superfusion with etidocaine, an agent causing increased leakage of stored transmitter, clorgyline abolished the appearance of DOPEG. Addition of semicarbazide in preparations treated with pargyline did not affect the efflux of deaminated and O-methylated deaminated metabolites. From the measurement of tissue VMA, it appeared that the efflux of VMA poorly reflects quick changes in the rate of its formation but that formation is abolished by pretreatment with pargyline. These experiments indicate that in the canine saphenous vein: (1) DOPEG is formed mainly in intraneuronal sites, while DOMA, MOPEG and VMA are formed extraneuronally; (2) VMA is retained in the tissue after its formation; and (3) the only subtype of MAO involved in the metabolism of 3H-noradrenaline released from adrenergic nerve endings can be classified as MAO-A.

The "half-saturating outside concentration" and the Vmax of the extraneuronal O-methylating system of the dog saphenous vein were determined in vitro for 3 catecholamines: isoprenaline, adrenaline and noradrenaline. Strips pretreated with 1 mmol/l pargyline were exposed to 30 mumol/l cocaine for 30 min before and during the 30 min incubation with the amines. Two methods were used to reach our aims: a) the classical one in which the 3H-O-methylated metabolites formed from a mixture of unlabelled and labelled amine were determined by using final concentrations of the substrate ranging from 0.2 and 12.8 mumol/l and b) an indirect one in which 0.2 mumol/l 3H-(+/-)-isoprenaline was used to assess the extraneuronal O-methylation of the tracer amine, and then those concentrations of unlabelled amines were determined which reduce the O-methylation of 3H-(+/-)-isoprenaline by 50% (IC50). The "half-saturating outside concentrations" and the Vmax obtained by the first method were: 1.3, 2.5 and 3.4 mumol/l and 241, 317 and 294 pmol/g/min for 3H-(+/-)-isoprenaline, 3H-(+/-)-adrenaline and 3H-(-)-noradrenaline, respectively. The IC50s obtained by the second method used were: 1.1, 0.6, 0.7 and 1.4 mumol/l for (+/-)-isoprenaline, (-)-isoprenaline, (-)-adrenaline and (-)-noradrenaline, respectively. It was observed that the contraction of the strips caused by adrenaline and noradrenaline distorted IC50 values. In the presence of 1 mumol/l phentolamine the IC50 for adrenaline and noradrenaline was about 2.5 times higher than in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

Spirally cut strips of human saphenous veins preincubated with 3H-noradrenaline were superfused in the presence of corticosterone and, unless stated otherwise, of cocaine or desipramine. Tritium overflow was stimulated electrically (2 Hz). Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential beta 2-adrenoceptor agonist procaterol concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a beta-adrenoceptor agonist with moderate preference for beta 1-adrenoceptors, was ineffective. The concentration-response curve of isoprenaline was shifted to the right by the nonselective beta-adrenoceptor antagonist propranolol and by the preferential beta 2-adrenoceptor antagonist ICI 118,551, but was not affected by the beta 1-selective antagonist atenolol. In experiments on strips preexposed to adrenaline 10 nmol/l (i.e. a concentration higher than that which normally occurs in vivo) for 32 min in the absence of cocaine or desipramine, the electrically evoked 3H overflow was not affected 12 and 44 min after withdrawal of adrenaline, irrespective of whether propranolol was absent or present in the superfusion fluid. In veins incubated with 3H-adrenaline, a considerable amount of the radioactivity was accumulated. During subsequent superfusion with 3H-adrenaline-free solution, electrical stimulation induced tritium overflow in a tetrodotoxin-sensitive manner. Propranolol failed to modify the evoked tritium overflow. It is concluded that the sympathetic nerve fibres of the human saphenous vein are endowed with facilitatory presynaptic beta 2-adrenoceptors. These receptors do not seem to play a substantial role in a local adrenaline (previously taken up)-mediated positive feedback loop regulating noradrenergic transmission, at least under the present in vitro conditions.

Strips of canine saphenous vein were loaded with 3H-noradrenaline (1.4 micrometer) and perifused with Krebs solution and either subjected to field stimulation or exposed to tyramine 40 micrometer. 3H, noradrenaline and its metabolites were determined in the perifusion fluid. Stimulation caused an increase predominantly in noradrenaline, followed by DOPEG, whereas tyramine release DOPEG in larger amounts than noradrenaline. Tyramine had more sustained effects than stimulation. Cocaine (1.6 micrometer) drastically reduced DOPEG efflux due to stimulation, but had no effects on the pattern of release by tyramine. It is concluded that tyramine releases noradrenaline which is deaminated before it reaches the synaptic gap, whereas after stimulation deamination of the transmitter occurs after re-uptake.