Livio Argiolas

Neuropeptide Y‐induced potentiation of noradrenergic vasoconstriction in the human saphenous vein: involvement of endothelium generated thromboxane

We investigated the potentiating effect of low concentrations of neuropeptide Y (NPY) on the vasoconstriction induced by transmural nerve stimulation (TNS) and noradrenaline (NA) in human saphenous veins. The effects of (i) endothelium removal; (ii) the addition of the NO pathway precursor L‐arginine; (iii) the ETA/ETB endothelin receptor antagonist Ro 47‐0203; (iv) the cyclo‐oxygenase inhibitor, indomethacin; (v) the selective thromboxane A2 (TxA2) receptor antagonists Bay u3405 and ifetroban, and (vi) the TxA2 synthase inhibitor, UK 38485, were studied in order to gain information about the mechanisms of NPY‐induced potentiation. Contractile response curves for TNS (0.5–8 Hz) and for exogenously administered NA (0.1–3 μM) were obtained in superfused saphenous vein rings. The contractions induced by both TNS and NA at all tested frequencies and concentrations, respectively, were significantly potentiated by 50 nM NPY in endothelium intact veins. Conversely, in endothelium‐denuded vessel rings the contractile‐response curves to TNS and NA overlapped both in the absence and presence of NPY, thus suggesting that a release of vasoactive substances from endothelial cells could account for the noradrenergic NPY‐induced potentiation. In vessels with intact endothelium, the potentiating action of NPY on TNS and NA was unaffected by the presence of high concentrations of the NO precursor L‐arginine (3–10 mM) or the non‐selective ETA/ETB endothelin receptor antagonist, Ro 47‐0203 (10 μM). These data indicate that the NPY‐induced effect does not involve either the endothelium‐derived vasodilator nitric oxide or the vasoconstrictor endothelin. Conversely, in the presence of the cyclo‐oxygenase inhibitor, indomethacin (30 μM), NPY failed to potentiate the vasoconstrictions produced by either nerve stimulation or by exogenous NA, thus providing evidence that arachidonic acid metabolites through the cyclo‐oxygenase pathway are mainly responsible for the potentiation evoked by NPY. When the TxA2 receptor antagonists, Bay u 3405 (1 μM) and ifetroban (1 μM) were added to the superfusing medium, NPY did not alter either the frequency‐ or the concentration‐response curves for either TNS or NA. Accordingly, both TNS‐ and NA‐induced contractions were not potentiated by NPY in the presence of the TxA2 synthase inhibitor, UK 38485 (10 μM). This clearly demonstrates the pivotal role of TxA2 in NPY‐induced potentiation. In superfused vein rings with endothelium, a subthreshold concentration (0.2 nM) of the TxA2 mimetic U 46619 potentiated both TNS‐ and NA‐induced vasoconstrictions. This potentiation was higher at low stimulation frequencies and low NA concentrations, and resembled that produced by NPY. Our results indicate that in the human saphenous vein NPY potentiates the contractions produced by sympathetic nerve stimulation acting at the postjunctional level, primarily on endothelial cells. In particular, the NPY‐induced release of a cyclo‐oxygenase metabolite, namely TxA2, may have a synergistic effect on the vasoconstriction induced by the noradrenergic mediator. Thus, such a mechanism may play a key role in the maintenance of the sympathetic tone of large human capacitance vessels.

Evidence for sympathetic neurotransmission through presynaptic N-type calcium channels in human saphenous vein.

1 The specific type(s) of voltage‐sensitive calcium channels (VSCCs) involved in sympathetic neurotransmission have not yet been characterized in human vascular tissues. We therefore examined the functional role of the N‐ and L‐type VSCCs in human saphenous veins. 2 Contractile response curves for transmural nerve stimulation (TNS) and for exogenously administered noradrenaline (NA) were obtained in superfused saphenous vein rings. The contractions induced by TNS, but not by NA, were inhibited by 1 μm tetrodotoxin and by 10 μm guanethidine. Both responses were substantially reduced by 1 μm phentolamine, indicating that the contractions evoked by TNS were mediated by endogenous NA released from noradrenergic nerves. 3 In the presence of 2 μm ω‐conotoxin GVIA (omega Conus Geographus toxin, fraction VI A; ω‐CgTx), a polypeptide with specific inhibitory activity on N‐ and L‐type calcium channels, the neurally evoked contractions were almost completely abolished. In contrast, the responses induced by exogenous NA were not affected by the neurotoxin, thus providing evidence of the exclusive presynaptic action of ω‐CgTx. 4 In the presence of the calcium antagonist verapamil (10 μm), which selectively blocks L‐type VSCCs, the contractions induced by both TNS and NA were diminished to the same extent, suggesting that the organic calcium blocker is active only at the postjunctional level. 5 It is concluded that N‐type calcium channels are the main pathway of calcium entry controlling the functional responses induced by activating sympathetic nerves; the role of L‐type channels appears to be limited to the postjunctional level, modulating smooth muscle contractions.

Mechanisms of pulmonary vasoconstriction and bronchoconstriction produced by PAF in the guinea-pig: role of platelets and cyclo-oxygenase metabolites.

1 The mechanisms of action of platelet activating factor (PAF) in the bronchial and cardiovascular systems have not yet been fully elucidated. In order to characterize better and to ascertain whether the effects of PAF in both these systems may be ascribed to the same mechanisms, we examined the actions of PAF in the heart‐lung preparation of guinea‐pig (HLP). The role of platelets and of cyclo‐oxygenase metabolites was investigated. 2 In HLPs perfused with autologous blood, bolus injections of PAF (4–32 ng) produced major effects at the pulmonary vascular and bronchial levels. Both dose‐related pulmonary vascular hypertension and bronchoconstriction produced by PAF were diminished to the same extent (46% and, respectively, 47%) when HLPs were perfused with a medium consisting of homologous red blood cells suspended in physiological solution containing 3.5% dextran (RBC). This suggests that the effects of PAF partially depend on the presence of formed elements. 3 When indomethacin (30 μm) was added to the perfusing blood, the dose‐response curve for the pulmonary hypertensive responses produced by PAF was strongly reduced (90%) in comparison to control preparations, whereas the bronchoconstrictor effects of PAF were only partially diminished (23%). These data constitute direct evidence that products of the cyclo‐oxygenase pathway exert a major role in the vascular, rather than in the bronchial actions of PAF. 4 In HLPs perfused with RBC containing indomethacin (30 μm), the pulmonary vascular hypertensive responses produced by PAF were almost completely abolished, thus indicating that cyclo‐oxygenase products from tissues are involved in these effects. Conversely, PAF administration continued to cause dose‐related bronchoconstrictor responses that were reduced only partially in comparison with HLPs perfused with RBC in the absence of the cyclo‐oxygenase inhibitor. This implies that PAF also has direct action on the bronchoconstriction evoked. 5 At the cardiac level, administration of PAF in HLPs perfused with blood caused a dose‐related increase in right atrial pressure accompanied by a decrease in left atrial pressure and cardiac output, which were completely suppressed or attenuated by the absence of formed elements and the addition of indomethacin. This suggests that the progressive heart impairment is secondary to the severe pulmonary hypertension induced by PAF. 6 The results of this study performed in the heart‐lung preparation of the guinea‐pig, which made it possible to simultaneously record cardiovascular and bronchial parameters, indicate that various components are involved in the responses produced by PAF. It is suggested that different mechanisms depending on the relative contribution of these components may account for the PAF‐induced effects at the pulmonary vascular and airway levels.

Nitric oxide-dependent and -independent modulation of sympathetic vasoconstriction in the human saphenous vein

The possible modulation by the endothelium of the contractile responses to sympathetic nerve stimulation was examined in isolated superfused human saphenous vein. Contractile response curves for transmural nerve stimulation and noradrenaline were higher in endothelium-denuded than in intact human saphenous vein rings. In vessels with endothelium, transmural nerve stimulation- and noradrenaline-induced contractions were unaffected by the cyclooxygenase inhibitor, indomethacin (10 microM), but were potentiated by the nitric oxide (NO) synthase inhibitor, L-N omega-nitro-L-arginine (L-NNA, 3 microM) even when combined with D-arginine (0.3 mM), but not with L-arginine (0.3 mM). As in the case of noradrenaline, contractile responses to 5-HT, but not to KCI, were enhanced by endothelium removal, L-NNA or L-NNA plus D-arginine, but were unaffected by L-NNA plus L-arginine. The guanylyl cyclase inhibitor, methylene blue (10 microM), potentiated both transmural nerve stimulation- and noradrenaline-induced contractions in endothelium intact rings, whereas it enhanced, although to a lesser degree, only the neurally evoked contractions in endothelium-denuded human saphenous vein. In the vessels without endothelium L-NNA failed to affect the vasoconstriction induced by both transmural nerve stimulation and noradrenaline. Our results suggest that at least two inhibitory factors are involved in modulating the sympathetic vasoconstriction in the human saphenous vein: (1) at a postjunctional level, NO, the release of which from endothelial cells is probably stimulated by the activation of specific receptors, and (2) at a prejunctional level, an unidentified vasodilator agent, which is unmasked by the removal of the endothelium layer and which is probably co-released along with noradrenaline, and which acts through the guanylyl cyclase pathway.

Cardiopulmonary effects of endothelin-1 in the guinea pig : role of thromboxane A2

Summary: The effects of endothelin-1 (ET-1) on the cardiovascular and bronchial systems were examined in a heart/lung preparation of guinea pig. The role of arachidonic acid metabolites through cyclo-oxygenase (COX) and lipoxygenase (LOX) pathways was investigated. Bolus injection of ET-1 (25–400 ng) caused dose-related bronchoconstriction, pulmonary vascular hypertension, and cardiac output reduction. When indomethacin (30 μM) or the thromboxane receptor antagonist Bay u 3405 (1 μM) were added to the perfusing blood, the cardiopulmonary effects of ET-1 were almost completely abolished. Conversely, the presence of the LOX inhibitor Bay x 1005 (10 μM) did not affect the ET-1 produced actions. We concluded that ET-1 exerts both bronchial and pulmonary vascular effects through an indirect mechanism. COX products, most likely thromboxane A2 but not arachidonic acid metabolites via the LOX pathway, make the major contribution to the bronchoconstrictor and pulmonary vascular hypertensive effects of ET-1.

Influence of morphine and cylazocine on the cortical epileptic foci in rabbits

The effects of morphine, cyclazocine and naloxone on penicillin- and strychnine-induced epileptic foci were studied in rabbits. The intracortical injection of penicillin (75, 150 and 300 units) elicited isolated spikes followed by repeated ictal events. The application of strychnine (0.062 and 0.125%) over the cortical surface of one side induced appearance of ipsilateral spiking spreading to the contralateral cortex. Administration of morphine (0.25-0.75 mg/kg i.v.) or cyclazocine (0.05-3.0 mg/kg i.v.) inhibited the occurrence or the duration of the EEG and motor manifestations induced by penicillin (75 and 150 units) and strychnine (0.062 and 0.125%), while it did not influence the effect of 300 units of penicillin. High doses of morphine (up to 10 mg/kg i.v.) failed to affect the epileptic responses to penicillin and strychnine and at the same time significantly reduced the pO2 in arterial blood. Naloxone per se potentiated the effects of the lower doses of penicillin and strychnine. Only at very high doses (20 mg/kg i.v.) displayed a weak antagonism towards the anticonvulsant effect of the two opiates. A full antagonism is only observed towards the effect of cyclazocine (2 mg/kg i.v.) administered after penicillin. Present data provide additional evidence of the heterogeneity of regulation by opioids of convulsive phenomena. One can hypothesize that the anticonvulsant effect of the two opiate agonists is mediated by naloxone-insensitive opiate receptors, while the proconvulsant-convulsant effect of naloxone might be related to an inhibition of GABA and glycine-mediated transmission.

Effects of dihydrotestosterone treatment on adrenal gland function and morphology in adult female guinea-pigs

The effect of chronic treatment of female guinea-pigs with dihydrotestosterone (DHT) on growth and function of the adrenal gland and, in particular, on the reticular zone is described. Two groups of 6 young adult, female guinea-pigs were treated with DHT (1 mg/kg dissolved in peanut oil and injected s.c.) for 30 and 60 days. Two other groups of animals, treated only with oil, were used as controls. At the end of treatment, animals were killed and adrenal glands were quickly removed. Plasma levels of pregnenolone, dehydroepiandrosterone (DHA) and its sulfate (DHA-S), 17 alpha-hydroxyprogesterone, androstenedione, testosterone, estradiol, 11-deoxycortisol, androstenedione, DHT and 3 alpha-androstanediol were determined by R.I.A. following celite microcolumn chromatography. Animals treated for 30 days showed only elevated DHT and 3 alpha-androstanediol plasma levels, whereas animals treated for 60 days also showed increased values of pregnenolone (251 +/- 62 vs 193 +/- 51 ng/dl; P less than 0.05), DHA-S (12,046 +/- 4110 vs 2780 +/- 888 ng/dl; P less than 0.001) and slightly increased values of DHA (110 +/- 31 vs 86.5 +/- 55.4). In the 30-day-treated animals no histological changes were observed, but in the 60-day-treated group the total size as well as cell volumes of the zona reticularis were significantly increased. Normal estrous cycles were observed in the 30-day-treated animals whereas the 60-day-treated animals showed a progressive acyclicity during the second month of treatment. These results indicate that in guinea-pigs, prolonged treatment with DHT induces a growth of the zona reticularis of the adrenal gland associated with increased levels of 5-ene steroids, particularly DHA-S. The mechanisms inducing these modifications are probably mediated by a DHT effect at the hypothalamic-pituitary level. A direct effect of DHT on the zona reticularis, however, cannot be excluded.

In vivo metabolism of fluorescent ceramide in central nervous system myelin of adult rats

The metabolism of sphingolipids in the central nervous system (CNS) has been studied in adult rats by intraventricular administration of fluorescent ceramide (CER). Rats were sacrificed at various time points post inoculation and the fluorescence of CER, cerebrosides (CB), sulfatides (SULF) and sphingomyelin (SPM) was determined in the CNS myelin and in the pellet, containing mainly microsomes, obtained by Norton myelin preparation. The incorporation of fluorescence was more in the pellet than in the myelin at all times studied. Initially the fluorescence present in the pellet was prevalently due to untransformed CER but an increase of fluorescent products with time was observed. CB was the main product up to 2 h post inoculation, then it decreased with concomitant increase of fluorescent SULF. In the myelin we did not observe differences in incorporation and transformation of fluorescent CER with time: CB was the main fluorescent product at all times studied. At 0.5 h post inoculation the fluorescence, observed by fluorescence microscope, was located in the cell lining the ventricles while after 24 h it appeared also in the paraventricular areas.

Thromboxane generation and pulmonary reactivity to arachidonic acid in heart-lung preparation of guinea-pig: Modulation by PCO2

A dose-related increase of pulmonary vasoconstrictive and bronchoconstrictive effects, as well as of the amounts in the perfusing fluid of TXB2, the stable metabolite of TXA2, was obtained through administration of arachidonic acid (AA) in normocapnic and deeply hypocapnic guinea-pig heart-lung preparations (HLPs) perfused with homologous red blood cells suspended in a modified Tyrode solution. Pulmonary hypertensive effects and the amounts of TXB2 detected in the perfusing fluid were reduced in hypocapnic preparations as compared with the normocapnic ones, while the bronchoconstrictive responses to AA were not affected by CO2 tension. It is concluded that: a) biosynthesis of TXA2 is reduced in hypocapnic group if compared with that observed in normocapnic one, b) the quantitative change of AA metabolism is responsible for hypocapnia reduction of pulmonary vasoconstrictive effects of AA, c) stability of bronchoconstriction due to AA infusions in normocapnic and hypocapnic HLPs might indicate an up regulation for TXA2 bronchial smooth muscle receptors by hypocapnia.