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8 janvier 2004
2001; 22; 1453-1457
Substance P and its transglutaminase-synthesized spermine derivative elicit yawning behavior via nitric oxide in rats
F Mancuso, R Porta, A Calignano, P Di Pierro, M Sommella, C Esposito
Department of Experimental Pharmacology, University of Naples, Italy


Introduction Substance P (SP), neurokinin A and neurokinin B are members of a family of structurally related mammalian neuropeptides, termed neurokinins (NKs), having excitatory effects on both central and peripheral neurons. NKs elicit various biological responses also in non neuronal tissues, including pain transmission, neurogenic inflammation, vasodilation, smooth muscle contraction, intestinal motility, as well as immune response. The physiological actions of NKs are exerted via three different receptors, named NK1 NK2 and NK3.
The three receptor sites, all belonging to the superfamily of G-proteincoupled receptors, are differently expressed in the nervous systern and peripheral tissues, one or more of them being responsible for distinct biological respons. SP exerts its physiological actions via the SP-preferring NK1 receptors which are highly expressed in different brain regions. The distribution of NK1 receptors in brain reflects the wide variety of behavioral changes after central administration of SP or selective NK1 agonists. The stimulation of NK1 receptors triggers several second messenger systems and distinct behavioral effects are observed in experimental animals. NK1 receptor has been cloned and species-related variations were observed in the primary sequence. Moreover, there is evidence for an NK1 subtype, which is distinct from the "classic" NK1 receptor present in some peripheral tissues and in the rat striatum coupled to distinct transducing systems.
SP is the most abundant NK in the mammalian brain and the distribution of NK1 receptors in the mammalian CNS has been investigated by using different techniques. NK1 receptors are abundant in the substantia nigra, striatum, nucleus accumbens, hippocampus, lateral nucleus of the hypothalamus and the nucleus of the tractus solitarius. There are immunochemical evidence indicating that almost 90% of dopaminergic neurons in the substantia nigra are immunoreactive to anti-SP receptor antibody, although the role of this neuropeptide in this area is still unclear.
In previous studies we demonstrated that transglutarninasesynthesized -y-(glutamyl5)spermine derivative of SP (Spm-SP) could be a useful tool in differentiating NK1 receptors. In fact, Spm-SP retains several NO-mediated biological activities evoked by the native SP, such as the ability to provoke rabbit aorta relaxation, to decrease rat arterial blood pressure and to inhibit collagen-induced platelet aggregation. In contrast, Spm-SP is completely ineffective to contract the smooth muscle isolated from different tissue preparations (stomach strips, duodenum, and ileal segments) of both gumea pig and rat, and it was almost completely unable to induce edema formation when injected in the rat paw. Moreover, Spm-SP was found completely unable to cause bronchoconstriction in guinea pig in vivo being unable to recognize NK1 receptors occurring in guinea pig bronchi, where NK1 receptors are pharmacologically distinct from those present in rat vascular system. Therefore, all these data suggest that Spm-SP could be an useful tool in differentiating the NOmediated effects of SP also in mammalian brain.
Those circumstances drove us to investigate the role of SP in the substantia nigra using its spermine derivative to investigate which type of NK1 receptor is involved in yawning event and to better clarify the role of the second messenger of this neuropeptide in this area. [...]
Discussion SP, one of the best known and the most abundant NK in the mammalian brain and peripheral neurons, exerts its physiological actions via the SP-preferring NK1 receptors which are highly expressed in different brain regions. Moreover, studies led to the proposal that two NK1 receptor subtypes exist in the brainstem, one recognized with high affinity by SP and the other one showing with high affinity for septide. Substantia nigra receives abundant SP innervations and recently SP receptor(s) have been found on dopamine neurons in pars compacta.
In agreement with this view, it has been reported that stimulation of oral movements after intranigral injection of a tachykinin NK1 receptor agonists is enhanced in rats after chronic treatment with neuroleptics. The involvement of SP in the regulation of dopamine effects it has been also shown in experimental model of catalepsy in which SP inhibited acetylcholine release from striatum. We also shown the regulatory effect of SP in locomotion and food intake, widely believed dopaminergic effect in rats. Here we have extended our prior observations on the regulatory role of SP in the nigrostriatal area in controlling the release of dopamine via NO.
Since we previously demonstrated that an enzymatically synthesized analog of SP, its -y-(glutamy 15 )spermine derivative, could be a selective SP agonist eliciting only the NO-mediated effects of the neuropeptide, it was of interest to investigate the effects of injecting the selective septide-sensitive NK1 receptor agonist Spm-SP into the nigrostriatal region of the rat brain on yawning. Yawning is a physiological behavior that occurs alone or associated with penile erection in mammals under different conditions and it is under the control of several neurotransmitters and neuropeptides at the central level.
The best known are dopamine, acetylcholine, serotonin, adrenocorticotropic hormone and oxytocin. Also neurotensin and luteinizing hormone-releasing factor are involved in this behavioral response. Experimental evidence has shown that NO, a molecule occurring in several tissues including the central nervous system, is involved at the paraventricular level in this symptomatology when its synthesis is induced by dopamine D2 receptor agonists. It has been suggested that NO may act as neurotransmitter and, because it readily diffuses across cell membranes, it might be not confined inside the cells in which it is produced.
The results of the experiments reported in the present paper indicate that yawning induced by intranigrostriatal injection of SP or Spm-SP reflects activation of NO-mediated NK1 receptors. In fact, as we previously demonstrated, Spm-SP retains only NO-mediated biological activities evoked by native SP. Moreover, the administration of L-NAME, a well known NO-synthase inhibitor, stereospecifically reduced in a dose related manner both SP and Spm-SP-induced yawning, whereas L-arginine pretreatment was shown to prevent: the effect of NO-synthase inhibitor on the drug-induced yawning.
Our finding strongly support the hypothesis that NO is involved in dopamine release. Furthermore, the experiments performed by using RP,67580, in which the yawning behavior was completely prevented, demonstrated the NK1 receptor involvement in the effect induced by both SP and Spm-SP. It is worthy to note that RP,67580, a well known NK1 antagonist receptor, had a high affinity for the septidesensitive sites in different tissues.
Since it is known that NO is involved in the induction of yawning by other neuropeptides and some neuronal links (i.e. dopamine/serotonine and dopamine/acetylcholine) we repeated the experiments in reserpinezed rats to establish a possible relationship between NO-mediated NK1 receptors and dopaminergic neurons.
The observed increasing of yawns when reserpine depletes the dopamine content clearly indicates that this behavior is dependent upon endogenous dopamine levels in the intranigrostriatal area of rat brain. Furthermore, the SP-induced yawning was unaffected by propanolol, prazosine, yohimbine or mianserine suggesting that neither cathecolamines or serotonine are involved in Spm-SP induced yawning.
These results are in line with the existence of SP receptors in dopamine neurons of substantia nigra pars compacta recently described and strongly suggest the role of NK1 receptor, probably the septide-sensitive type, in controlling dopamine release via NO.

Furthermore, interaction between the meso-striatal dopamine system and NKs have been observed with a number of interdisciplinary approaches and a neuronal link between dopamine and acetylcholine systems has also been well established. In fact, a muscarinic receptor antagonist able to cross the blood-brain barrier, as scopolamine, prevents the yawning behavior induced by dopamine D2 receptor agonists and oxitocin. We demonstrated that SP and Spm-SP yawning-inducing effect was prevented in a dosedependent manner by using scopolamine, but not methylscopolamine. All the above considerations thus suggest that a dopamine/acetylcholine link is involved in the yawning response induced via NO by both SP and Spm-SP activation of NK1 receptors at the nigrostriatal level.