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Neuropeptide Y (NPY) is now well known to be co-localized in perivascular sympathetic nerve fibres and co-released with noradrenaline (NA) from sympathetic nerve endings. At the prejunctional level, it reduces the release of NA (for reviews, Edvinsson et al., 1987; Potter, 1988), whereas at the postjunctional level, at relatively high concentrations, probably via activation of NPY1 receptors (Oellerich & Malik, 1993; Westfall et al., 1995) it is a vasoconstrictor in most but not all vessels (Edvinsson et al., 1984; Wahlenstedt et al., 1985). At lower concentrations, NPY can increase the vasoconstriction induced by various contractile agents (Edvinsson et al., 1984; Andriantsitohaina & Stoclet, 1988; Macho et al., 1989; Westfall et al., 1995) or by perivascular nerve stimulation (Wong-Dusting & Rand, 1988; Vu et al., 1989; Saville et al., 1990).
However, NPY-induced vasoconstriction potentiation has been observed to be a tissue- and species-dependent effect, and discrepant results have also been obtained with regard to its mechanism of action. The responses to NA were enhanced in rabbit gastroepiploic and femoral arteries, but not in veins (Edvinsson et al., 1984), and in rat mesenteric arteries, but not in femoral veins (Pernow et al., 1986). However, NPY potentiating effects on both transmural nerve stimulation (TNS) and exogenous NA were observed in canine saphenous veins (Hieble et al., 1989). Concerning the mode of action of NPY, differences have been found with respect, for example, to the role of endothelium. In rat isolated tail and mesenteric arteries, NA-induced contractile responses were potentiated by NPY in the absence of endothelium (Gustafsson & Nilson, 1990; Small et al., 1992). Conversely, in the rabbit isolated perfused ear artery and in canine saphenous vein, an intact endothelium was required for the potentiation of NA- and TNS-mediated contractions by NPY (Daly & Hieble, 1987; Hieble et al., 1989). In addition, in bovine isolated retinal arteries, the release of an endothelium-derived contracting factor has been recently described to account for the potentiating effect of NPY in the proximal, but not the distal, part of the vessel (Prieto et al., 1995).
Therefore, we deemed it interesting to verify whether NPY was able to enhance the vascular autonomic tone of large human venous capacitance vessels, and to study the role of the endothelium in the phenomenon. Thus with regard to the mechanisms of this NPY-induced effect, we hypothesized that either (i) the inhibition of the release of a relaxing factor, or (ii) the stimulation of the release of a contractile factor by the endothelium and/or the muscular layer might be responsible for the NPY-potentiating effect.
The possibility that NPY could inhibit the release of an endothelium-derived relaxing factor (particularly NO) seemed reasonable in view of the observations that in human omental arteries (Aldasoro et al., 1993) and saphenous veins (Fabi et al., 1996) the contractile responses to both TNS and NA were enhanced by endothelium removal or by the presence of the NO synthase inhibitors. On the other hand, we could not exclude the speculation that the ability of NPY to potentiate the responses to nerve stimulation and exogenous NA might be explained through the release of vasoconstrictor substance(s), which can synergistically act with the noradrenergic mediator at the postjunctional level. Indeed, it is well recognized that different stimuli induced endothelial and/or smooth muscle cells to release different contractile factors. Endothelin (Furchgott & Vanhoutte, 1989; Yang et al., 1990) and/or prostanoids, particularly thromboxane A2 (TxA2; Chester et al., 1993; Cocks et al., 1993), are the endogenously released substances most likely to act as secondary mediators in vasoconstriction.
Therefore, the main aims of our work were (1) to investigate whether NPY was able to enhance the frequency- and concentration-response curves to TNS and to NA, respectively, in human saphenous veins, (2) to verify the role of the endothelium in such potentiation, and (3) to characterize the endothelium-derived factor, if any, involved in the phenomenon.
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The present study demonstrated firstly that NPY potentiates the contractions elicited by the transmural stimulation of sympathetic nerves in human saphenous veins in vitro. This effect is most likely due to an action of the neuropeptide at the postjunctional sites to increase the vascular reactivity to the neurotransmitter released from the sympathetic nerve fibres since, in the same experimental vessels, NPY also potentiated the contractions induced by exogenously administered noradrenaline (NA). This confirms the data previously obtained in several isolated blood vessels from various experimental animals (Pernow et al., 1986; Wong-Dusting & Rand, 1988; Hieble et al., 1989; Saville et al., 1990). In addition, with regard to the mode of action of NPY in this phenomenon, our results are primarily in favour of the view that NPY-induced potentiation in human saphenous veins involves the activation of an endothelium-dependent mechanism.
As mentioned in the Introduction, the role of the endothelium on NPY-induced potentiation of noradrenergic vasoconstriction in isolated blood vessels is controversial. In the majority of the studies, carried out on several isolated mammalian vessels, the potentiating effects of NPY on TNS-and/or NA-induced contractions appear to be independent of the presence of an intact endothelium (Gustafsson & Nilsson, 1990; Adamsson et al., 1992; Small et al., 1992), but few studies have described an endothelium-dependent mechanism as well (Daly & Hieble, 1987; Hieble et al., 1989). Some doubt could arise about the role of endothelial cells in the phenomenon in view of the contradictory observations of an endothelium-dependent (Daly & Hieble, 1987) and -independent (Budai et al., 1989; Gustafsson & Nilson, 1990) NPY potentiation obtained by different authors in the same type of blood vessel, i.e. rabbit ear artery. Nevertheless, the recent results by Prieto et al., (1995) on the bovine retinal artery-which suggest that NPY triggers the release of an endothelium-derived contractile factor which facilitates the NA-induced contraction only in the proximal, but not in the distal, part of the artery-provide a reason for the contrasting results obtained in the rabbit ear artery, as well as confirming the role of the endothelium in NPY potentiation. In addition, an endothelium-dependent potentiation by NPY has also been described in a canine large capacitance vessel, i.e. the saphenous vein (Hieble et al., 1989). Thus, in agreement with these latter data, our results provide evidence that in human saphenous veins, the presence of an intact endothelium is a requirement for the NPY-induced potentiation of noradrenergic contractions.
As far as the mechanisms underlying the potentiating effect of NPY are concerned, most of the studies on the action of NPY have been performed on smooth muscle cells, supporting the theories that NPY may potentiate vasoconstriction by inhibiting adenylyl cyclase activity (Lundberg et al., 1988) or by promoting a G-protein-mediated calcium entry with increased accumulation of inositol triphosphate (Andriantsitohaina et al., 1990; 1993; Duckles & Buxton, 1994). Conversely, with regard to the mode of action of the neuropeptide in the endothelium-dependent potentiation, as far as we know, few data on the role of cyclo-oxygenase products and, by the way, with opposite results in different vessels, have been obtained (Hieble et al., 1989; Prieto et al., 1995). Therefore, as it is now well recognized that endothelial cells play a primary role in the regulation of the vascular function, mainly due to their capacity to produce strong vasodilator or contracting substances in response to a variety of stimuli (for review, see Li et al., 1994), we felt it reasonable to hypothesize that NPY potentiates the noradrenergic contractions of human saphenous veins either (i) by blocking the release of vasodilator substances which could modulate the contractions elicited by the sympathetic mediator or (ii) by producing contractile factors which act synergistically with the neuromediator.
The hypothesis that an NPY-induced inhibition of the nitric oxide (NO) pathway in the endothelial cells may be responsible for its potentiating effect on the noradrenergic contractions come from our previous observations on human saphenous veins, in which the same protocols were used (Fabi et al., 1996). In those experiments, the NO synthase inhibitor Nω-nitro-L-arginine (L-NOARG), potentiated the sympathetic contractions of vessels through an endothelium-dependent mechanism, probably blocking the noradrenaline-induced release of NO from the endothelial cells. This hypothesis was confirmed by the fact that the combined presence of L-NOARG and the NO precursor, L-arginine, did not cause any potentiation of the sympathetic contractions. Thus, our present finding that L-arginine at a very high concentration was not able to counteract the NPY-induced potentiation, reasonably suggests that an interference of NPY with the NO pathway or, more precisely, an L-NOARG-like action of NPY, is not the major mechanism of action of the neuropeptide.
A great deal of evidence is now available which demonstrates that in addition to mediating relaxation, the endothelium can also release contracting factors. In rat and canine basilar arteries, endothelium-dependent contracting factors have been suggested to facilitate the contractions elicited by various agonists (Katusic et al., 1988; Descombes et al., 1993). Moreover, it has been found that in certain large cerebral vessels and peripheral veins the normal endothelium has the propensity to release vasoconstrictor substances (Li et al., 1994). Among such endothelium-derived contracting factors, endothelins and cyclo-oxygenase metabolites appeared to us the most probable candidates to be involved in NPY-induced sympathetic potentiation. Indeed, endothelins, the family of peptides with 21 amino acids, which have potent vasoconstrictor properties (Yanagisawa et al., 1988; Inoue et al., 1989), have also been shown at subthreshold concentrations to potentiate the contractions elicited by NA in human arteries (Yang et al., 1990). However, our experiments in the presence of the ETA/ETB receptor antagonist Ro 47–0203, (Clozel et al., 1994) did not favour a major role of endothelins in the phenomenon. Conversely, the inability of NPY to potentiate noradrenergic contractions in the presence of indomethacin clearly indicates the involvement of cyclo-oxygenase metabolites in NPY potentiation.
The capacity of smooth muscle cells to produce contractile metabolites from arachidonic acid through the cyclo-oxygenase pathway, TxA2 in particular, is associated in canine and rat veins with the contractions produced by various peptide autacoids, like bradykinin (Aksoy et al., 1990; Campos & Calixto, 1994; Marsault et al., 1997), but controversial data exist on the role and the production of TxA2 from endothelial cells. Indeed, this potent vasoconstrictor substance is usually not considered the predominant endothelium-derived contractile factor (Furchgott & Vanhoutte, 1989), probably because, in certain blood vessels, the endothelium-dependent contractions evoked by various agonists are sensitive to cyclo-oxygenase inhibitors, but not abolished by TxA2 synthase blockers (Furchgott & Vanhoutte, 1989; Vanhoute et al., 1991). However, some data exist on the production of TxA2 from endothelial cells. The endothelium of rabbit pulmonary artery produced TxA2 both spontaneously and after arachidonic acid stimulation (Buzzard et al., 1993). In addition, contractions elicited by certain agonists in the canine and rat basilar artery (Katusic et al., 1988; Descombe et al., 1993) and in the rabbit intrapulmonary artery (Altiere et al., 1986) have been shown to cause endothelial TxA2 production, that contributes to the contractile response. Finally, as Lin et al., (1993) described the formation of TxA2 by the endothelium in the human internal mammary artery and, Yang et al., (1991) suggested that the production of TxA2 modulates the endothelium-dependent relaxation by acetylcholine in human saphenous veins, we deemed it reasonable to verify whether endogenously produced TxA2 may be involved in the potentiation by NPY.
This hypothesis was investigated in two different ways. Firstly, we tested the effects of two chemically different receptor antagonists, namely Bay u3405 (McKenniff et al., 1991; Norel et al., 1991) and ifetroban (Ogletree et al., 1993). Next, we inhibited the formation of endogenous TxA2 by perfusing the tissues with the TxA2 synthase inhibitor, U.K. 47–0203 (Parry et al., 1982). The finding that NPY-induced potentiation was inhibited by TxA2 receptor antagonists and the TxA2 synthase inhibitor in the same way as it was by indomethacin strongly supports the notion that, among the endogenously produced cyclo-oxygenase metabolites, TxA2 plays a substantial role in the NPY-induced potentiation of the noradrenergic contractions.
To give further support to this speculation, of particular relevance are the results of our experiments with U 46619, showing that threshold concentrations of the TxA2 stable agonist that did not elicit significant contractions by themselves, were capable of potentiating the contractions induced by both sympathetic stimulation and exogenous NA. In addition, this augmentation mimicked in extent and shape the potentiation produced by NPY in these vessels when the same experimental procedure and protocol was utilized. Indeed, as shown in Figure 8, (a) the percentage potentiating effect of both U 46619 and NPY on TNS and NA was higher at lower stimulation frequencies and lower NA concentrations and (b) the augmentation induced by U 46619 at all frequencies and concentrations tested completely overlapped the potentiation produced by NPY (percentage potentiation calculated on the data of Figure 7 and Figure 1; F= 0.08 and F= 0.10 for TNS and NA, respectively; P > 0.05 for both). All in all, our results suggest that endogenously produced TxA2, acting synergistically with the noradrenergic mediator, enhances the responses to sympathetic stimulation in human saphenous veins. This action of TxA2 may give some further insight into the mechanisms of vasospastic diseases, suggesting that not only the well-known platelet-derived, but also the vascular, release of TxA2 can contribute to the increase in the contractile responses to noradrenergic mediators in a human large capacitance vessel.
Figure 8. Comparison of the potentiating effects of 50 nM NPY (data from Figure 1) with those induced by 0.2 nM U 46619 (data from Figure 7) on the frequency- and concentration-response curves to transmural nerve stimulation (a) and noradrenaline (b) in superfused human saphenous vein rings with intact endothelium. The augmentations induced by NPY overlapped those induced by the thromboxane agonist (P > 0.05 for both transmural nerve stimulation and noradrenaline). Potentiation was calculated as the percentage increase of the second series of contractions vs the corresponding contractile responses of the first series. Values are means and vertical lines show s.e.mean.
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Taking into account all our findings, we hypothesize that, in human saphenous veins, NPY potentiates the sympathetic vasoconstriction through a postjunctional action, by stimulating the release of TxA2 from endothelial cells. Thus, given that it has been demonstrated that NPY, at a higher concentration than we used, also induces contractions in human saphenous veins without endothelium in vitro (Luu et al., 1992), our data further illustrates the complex interactions NPY has with perivascular neuroeffector mechanisms and show the modes by which NPY may also contribute to the sympathetic enhancement of the tone of venous capacitance vessels.