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.
