Franca Mancuso

Endogenous nitric oxide modulates morphine-induced changes in locomotion and food intake in mice

Opioids increase the dopaminergic turnover in nucleus striatum and nucleus accumbens of mice, causing behavioural changes such as increased locomotion and food intake. We have now shown that L-arginine administration increases morphine-induced locomotion and changes in food intake in mice. D-Arginine had no effect, suggesting a stereospecific mechanism. Furthermore NG-nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, reduced the morphine-induced effects. These results suggest that endogenous nitric oxide could play a role in the modulation of dopaminergic effects elicited by morphine.

Neurokinin Receptors Could Be Differentiated by Their Capacity to Respond to the Transglutaminase-Synthesized γ-(Glutamyl5)Spermine Derivative of Substance P

Abstract: Four different γ‐(glutamyl5)amine derivatives of substance P (SP) were synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The 1,3‐diaminopropane, spermidine, spermine (Spm), and monodansylcadaverine adducts of the neuropeptide were purified by HPLC on a reversed‐phase column and characterized by fast atom bombardment mass spectrometry. The γ‐(glutamyl5)Spm derivative of SP (Spm‐SP) was found to be able, like the parent neuropeptide, to provoke rabbit aorta relaxation, to decrease rat arterial blood pressure, and to inhibit collagen‐induced platelet aggregation. Unlike SP, only a weak inflammatory response was observed when Spm‐SP was injected in the rat hind limb. All these effects were found to be prevented by Nω‐nitro‐l‐arginine methyl ester, a well‐known nitric oxide synthesis inhibitor. In contrast, Spm‐SP was completely ineffective in contracting guinea pig ileal segments, thus confirming our preliminary observations indicating that Spm‐SP does not evoke SP‐like spasmogenic effects on isolated smooth muscle preparations. The specificity of the effects due to the selective introduction of a Spm moiety at the glutamine5 level was demonstrated by the SP agonist pharmacological profile of the other γ‐(glutamyl5)amine derivatives tested. These results suggest that neurokinin receptors could be differentiated by their capacity to respond to Spm‐SP.

Endogenous nitric oxide modulates behavioural effects elicited by substance P in rat

Several studies have shown that the undecapeptide, substance P, alters behaviour following central or peripheral administration in the rat. Here we report that L-arginine administration increases substance P-induced locomotion and changes in food intake in rats. NG-Nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, reduces substance P-induced effects. These results suggest that endogenous nitric oxide plays a role in the modulation of the catecholaminergic effect of substance P on motor behaviour. They also clarify the mechanism underlying food intake induced by substance P.

Substance P inactivation by transglutaminase in vitro

Gamma(glutamyl5)spermine derivative of substance P (Spm-SP) was synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The spermine adduct of the neuropeptide was purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The biological activities of Spm-SP were tested by assaying, in comparison with substance P, its ability to induce both the contractions of smooth muscle in vitro and the edema formation in vivo. Spm-SP was shown not to elicit contractile responses in the isolated rat stomach strip and duodenum and not to antagonize the spasmogenic effect evoked by the native neuropeptide. Furthermore, Spm-SP was unable, when administered into rats by plantar injection, either to provoke an acute inflammatory response in the hind limb or to antagonize the edema formation induced by a concurrent administration of substance P. These results indicate that the introduction of a large size hydrophilic moiety at the glutamine5 level negatively affects the ability of the neuropeptide to bind to its receptor(s), thus supporting the view that the hydrophobic middle portion of substance P plays a key role in receptor recognition.

Substance P and its transglutaminase-synthesized spermine derivative elicit yawning behavior via nitric oxide in rats.

Previously, we showed that intranigrostriatal injection of substance P (SP) cause behavioral changes in rats. Those effects, such as locomotion and food intake, resulted related to catecholamines release modulated by nitric oxide [18]. Here we report that intranigrostriatal injection of SP elicited yawning in rats. Moreover, since in previous studies we demonstrated that transglutaminase-synthesized gamma-(glutamyl5)spermine derivative of SP (Spm-SP) could be a useful tool in differentiating NK1 receptors [5,19,26], we reports the effects of injecting the selective septide-sensitive NK1 receptor agonist Spm-SP into the nigrostriatal region of the rat brain on yawning. The administration of L-N(omega)-nitroarginine methyl ester, a NO-synthase inhibitor, stereospecifically reduced in a dose related manner both SP and Spm-SP-induced yawning. In contrast, L-arginine pretreatment prevented the effect of NO-synthase inhibitor. Moreover, the NK1 antagonist RP,67580 blocked yawning behavior induced by both SP and Spm-SP, whereas the pretreatment with systemic reserpine determined its increase. The administration of NO-synthase inhibitor resulted ineffective in reducing SP and Spm-SP-induced yawns in reserpinized rats. Finally, yawns elicited by SP or Spm-SP were blocked when rats were treated with scopolamine but not with methylscopolamine. These results indicate that yawning induced in rats by SP injection is dependent upon endogenous dopamine levels in brain nigrostriatal area. Moreover, we demonstrate, by using Spm-SP, that septide-sensitive NK1 receptor are specifically involved in yawning behavior.

In vivo and in vitro inhibition of platelet aggregation by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

In summary, the present study documents that platelet aggregation triggered by thrombin, ADP, collagen and PAF both in vivo and in vitro, was prevented by SV-IV in a dose-dependent manner. Only platelet aggregation by AA was not affected by the protein, thus suggesting a possible involvement of PLA2 inhibition in the molecular mechanism at the basis of SV-IV anti-thrombotic effect.

Inhibition of zymosan-induced air-pouch inflammation by rat seminal vesicle protein and by its spermidine derivative

The anti-inflammatory effect of one of the major proteins secreted by rat seminal vesicles (SVIV) and of its spermidine derivative (Spd2-SVIV) was evaluated by measuring polymorphonuclear leukocyte migration, protein release, platelet-activating factor (PAF) and prostaglandin E2 levels in the mouse air-pouch exudate following zymesan treatment. Both proteins were found to markedly reduce dose dependently PAF and prostaglandin E2 levels in the exudate as well as the other parameters. Concurrent injection of either arachidonic acid or PAF, directly into the pouch, significantly counteracted the anti-inflammatory effect of SVIV and of its polyaminated derivative. These results support the notion that the molecular mechanism of the anti-inflammatory activity of SVIV and Spd2-SVIV is linked to the inhibition of both phospholipase A2 and acetyl:lyso-PAF acetyltransferase.

Biological activities of a major protein secreted from the rat seminal vesicles after structural modification catalyzed by transglutaminase in vitro

The immunosuppressive, anti-inflammatory and anti-thrombotic properties of SV-IV, a major protein secreted from the epithelium of rat seminal vesicles, were investigated after transglutaminase-catalyzed covalent incorporation of two molecules of spermidine (Spd) into the protein at the level of Gln-9 and Gln-86. The modified molecular form of the protein (Spd2-SV-IV) showed a more marked inhibitory activity on Con A-induced lymphocyte blastogenesis in comparison with the native protein, whereas no differences in the ability to inhibit the mixed lymphocyte reaction and to decrease the rat epididymal sperm immunogenicity were found between modified and native SV-IV. Spd2-SV-IV was also less effective than native SV-IV to inhibit platelet aggregation induced in vivo by different thrombogenic agents. In contrast, superimposable inhibitory tracings were observed in the in vitro platelet aggregation experiments performed with the two different molecular forms on the protein. Finally, Spd2-SV-IV was shown to retain unchanged the anti-inflammatory activity of native SV-IV.

Involvement of NK receptors and β-adrenoceptors in nitric oxide-dependent relaxation of rabbit aorta rings following electrical-field stimulation

Electrical-field stimulation caused an endothelium-dependent relaxation in rabbit aorta rings precontracted by phenylephrine. The relaxation was reduced in a dose-dependent manner by morphine, benzalkonium, [D-Pro2,D-Trp7,9]substance P and an beta-adrenoceptor antagonist, propranolol. The vasodilatation was enhanced by superoxide dismutase and abolished by haemoglobin and NG-monomethyl-L-arginine. The inhibitory effect of NG-monomethyl-L-arginine was reversed by L-arginine, the precursor of nitric oxide biosynthesis, but not by its enantiomer, D-arginine. These data show that the electrically induced relaxation is independent on nitric oxide released by NK receptors and beta-receptors. Moreover, morphine, by reducing substance P release, decreased the magnitude of electrically induced relaxation, suggesting an indirect role of opioids in the regulation of the peripheral circulation through the control of nitric oxide release. Furthermore our observations confirm the hypothesis that subtypes of beta-adrenoceptors releasing nitric oxide participate in the regulation of vascular tone.

Adenosine release in morphine-induced hypotension in rats

1. Following intravenous administration of morphine.HCl a reduction in mean arterial blood pressure (MABP) was produced, quaternary morphine analogue was ineffective. 2. Theophylline and 8-phenyltheophylline administration reduced morphine-induced hypotension. 3. A2 adenosine receptor agonist caused an hypotension while A1 adenosine receptor agonist was ineffective. 4. L-NG-Mono-methylarginine administration reduced the hypotensive effect of exogenous A2 agonist while it was ineffective on morphine-induced hypotension. 5. Morphine-induced hypotension was increased by pretreatment with dipyridamole, whereas tetrabenazine abolished it. 6. The present study is consistent with previous reports on the central hypotensive action of morphine and propose a role for adenosine release in morphine-induced hypotension.