N-terminal Fragment Substance P Research Articles

Investigation of the metabolic biotransformation of substance P in liver microsomes by liquid chromatography quadrupole ion trap mass spectrometry

Substance P (SP) belongs to the tachykinin family and plays an essential role in pain transmission and in neurogenic inflammation. It can be detected in the central and peripheral nervous systems. The objectives of this study were to establish SP metabolic stability in liver microsomes in three species (rat, mouse and human), and identify and characterize SP metabolites by LC-MS/MS. Endogenous peptide metabolism is not well documented and this is particularly true for neuropeptides participating in neurogenic inflammation. In vitro, T(1/2) results in pooled liver microsomes were 9.2, 5.6 and 18.6 min for rat, mouse and human liver microsomes, respectively. Five major SP metabolites were identified and quantified, including C-terminal SP fragments SP(3-11) , SP(5-11) , SP(6-11) , SP(8-11) as well as N-terminal fragment SP(1-7) . The results suggest significant differences between species in SP metabolite concentrations. Consequently, the metabolic profile of each species is distinctive and may have a significant impact on biomolecular mechanisms involved in specific pathophysiological changes.

Injection of substance P (SP) N-terminal fragment SP 1–7 into the ventral tegmental area modulates the levels of nucleus accumbens dopamine and dihydroxyphenylacetic acid in male rats during morphine withdrawal

The biologically active substance P (SP) N-terminal metabolite SP 1–7 has been reported to modulate several neural processes such as learning, locomotor activity and reaction to opioid withdrawal. Although all these processes are believed to be associated with dopaminergic transmission no evidence of an interaction between SP 1–7 and dopamine in the case of morphine withdrawal has so far been reported. Therefore, in this work we applied in vivo microdialysis to investigate the effect of SP 1–7 injection into the ventral tegmental area on dopamine release in nucleus accumbens of male rats during naloxone precipitated morphine withdrawal. The result showed that the heptapeptide enhances dopamine release and also elevates the level of the dopamine metabolite dihydroxyphenylacetic acid in this brain area. It was suggested that the observed action of the SP fragment on the dopamine system represents the underlying mechanism for a previously observed ability of SP 1–7 to counteract the aversion response to morphine withdrawal.

Purification of Substance P endopeptidase activity in the rat ventral tegemental area with the Äkta-Purifier chromatographic system

The new chromatographic system Äkta-Purifier 10 (Amersham–Pharmacia Biotech), scaled for preparative HPLC, was used for the purification of Substance P (SP) endopeptidase activity in the ventral tegemental area (VTA) of the rat brain. SP endopeptidase previously identified and purified from human cerebrospinal fluid has been found to degrade the neuroactive peptide SP in a specific pattern. In this study we have recovered SP endopeptidase from the rat VTA following a purification scheme involving homogenization (ultrasonication) and extraction of the excised tissue, size-exclusion chromatography (Superdex 75 HR), and ion-exchange chromatography (Resource Q). In this way we were able to achieve a purification factor of almost 7500, based on specific activity. The obtained SP endopeptidase activity, was then subjected to characterization with regard to inhibition profile. The enzyme activity was monitored by following the conversion of SP to its N-terminal fragment SP(1–7) using a radioimmunoassay, specific for the heptapeptide product. On basis of inhibition profile it was possible to discern two different SP endopeptidase-like activities, one sensitive toward the protease inhibitor phosphoramidon (preparation A), and another non-sensitive to phosphoramidon or captopril (preparation B). The molecular masses of preparations A and B, as derived from sodium dodecyl sulfate–polyacrylamide gel electrophoresis, were found to be 90 000 and 76 000, respectively. Our data suggest that the purified phosphoramidon sensitive endopeptidase activity may be an enzyme that plays a major role in the conversion of SP to its bioactive fragment SP(1–7) in the rat VTA. This is likely to be identical to the previously known neutral endopeptidase (EC 3.4.24.11). However, this study also demonstrates the existence of a distinct endopeptidase activity with properties in agreement with rat spinal cord SP endopeptidase. In the context of previously shown altered levels of SP(1–7) in the VTA during morphine withdrawal both purified enzyme activities may turn out to be responsible.

Intracerebroventricular injection of the N-terminal substance P fragment SP 1–7 regulates the expression of the N-methyl- d-aspartate receptor NR1, NR2A and NR2B subunit mRNAs in the rat brain

Studies have implicated substance P (SP) in the regulation of affective behaviour, memory function, pain influx, stress and opioid reward. All these dimensions are known to involve glutamate transmission mediated through the N-methyl- D-aspartate (NMDA) receptor. The SP N-terminal fragment SP 1–7 is shown to share some but oppose other effects of the parent compound. We have examined the effect of intracerebroventricular injections of SP 1–7 on the expression of the NMDA receptor subunits NR1, NR2A and NR2B mRNAs in the spinal cord and in discrete areas of the male rat brain. The results indicated that the heptapeptide induced a dose-dependent upregulation of the NR2A transcript in hippocampus, periaqueductal grey and ventral tegmental area, already within a few hours. The level of the NR2B mRNA was increased in hippocampus and nucleus accumbens. The expression of the transcript of the NR1 was enhanced in hippocampus and nucleus accumbens but attenuated in spinal cord. The observed effects of the SP 1–7 fragment are in agreement with what could be expected from the known effects of the heptapeptide on various behaviours involving glutamate transmission.

Anabolic-androgenic steroids affect the content of substance P and substance P 1–7 in the rat brain

The effects of intramuscular (i.m.) injections of nandrolone decanoate (15 mg/kg/day), an anabolic-androgenic steroid, on the levels of substance P (SP) and on its N-terminal fragment SP 1–7 were examined in the male rat brain by radioimmunoassay. The results demonstrated that the SP immunoreactivity in amygdala, hypothalamus, striatum, and periaqueductal gray was significantly enhanced, whereas the concentration of the N-terminal fragment SP 1–7 was enhanced in the nucleus accumbens and in periaqueductal gray. In the striatum the steroid induced a decrease in the content of SP 1–7. The relevance of these peptides in connection with anabolic-androgenic steroid-induced aggression is discussed.

Alteration in the brain content of substance P (1–7) during withdrawal in morphine-dependent rats

Previous studies have shown that substance P (SP) may modulate the abstinence reaction to opioid withdrawal. Its N-terminal fragment SP 1–7 may inhibit the intensity of the withdrawal reactions in morphine dependent mice. This study was designed to determine whether the endogenous concentrations of the SP 1–7 fragment in the brain are affected during naloxone-precipitated withdrawal in the male rat. The amounts of the peptide was assessed by a specific radioimmunoassay in extracts of discrete brain regions (including the cerebral cortex, hippocampus, hypothalamus, nucleus accumbens, striatum, substantia nigra, ventral tegmental area and the spinal cord) during morphine tolerance and withdrawal. The results indicated that the concentrations of SP 1–7 were significantly elevated in the ventral tegmental area both in morphine tolerant rats and during naloxone-precipitated withdrawal. During morphine withdrawal significant increases in the peptide concentration were also observed in the hypothalamus and the spinal cord. It was concluded that the enhanced content of SP 1–7 may also indicate the involvement of the SP system during opioid withdrawal in the rat. The enhanced production of SP 1–7 may reflect an increased release and/or metabolism of SP, which, in turn, counteracts the withdrawal.

Aversive effects of the C-fragment of Substance P in the dorsal periaqueductal gray matter.

There has been an increasing interest in the role of neuropeptides in the integration of brain functions. Besides the well-known positive-reinforcing effects of Substance P (SP) in prosencephalic regions, a role of this neuropeptide in the generation of aversive states in mesencephalic structures has also been envisaged. Evidence from a previous study suggests an involvement of SP in the neural substrates of aversion in the dorsal periaqueductal gray matter (DPAG). In the present study, we investigate whether N- and C-terminal fragments of Substance P are responsible for the effects produced by microinjections of SP into the dorsal periaqueductal gray. The results show that SP and its C-terminal fragment SP(7-11) produced a behavioral activation with increases in locomotor activity, grooming, and rearings, while the N-terminal fragment SP(1-7) produced only an increase in vertical exploratory activity. The effects were more pronounced with intermediate doses of SP and its C-fragment, confirming the characteristic bell-shaped dose-effect function of this neuropeptide. The proaversive effects observed with DPAG microinjections of these neuropeptides in the present study gain further relevance when combined with previous reports showing unconditioned and conditioned aversive effects following DPAG microinjections of SP in the place aversion and the elevated plus maze tests, two widely used animal models of anxiety. These results confirm previous data showing that SP has a modulatory role in the DPAG and that its effects are probably due to its C-terminal fragment.

The substance P fragment SP-(7-11) increases prostaglandin E2, intracellular Ca2+ and collagenase production in bovine articular chondrocytes

Substance P (SP) is found in increased concentrations in inflamed joints and is believed to play a role in joint pathology. Culture of bovine articular chondrocytes with SP or with the related mammalian tachykinins neurokinin A or B (NKA or NKB) produced no effect on prostaglandin E2 (PGE2) or collagenase production. However, the C-terminal fragment of SP, SP-(7-11), increased PGE2 and collagenase production at concentrations greater than 1 microM. The N-terminal fragments SP-(1-4) and SP-(1-6) had no effect on PGE2 or collagenase production. In addition, SP-(7-11), but not intact SP, SP-(1-4), SP-(1-6), SP-(8-11) or SP-(9-11), nor the tachykinins NKA and NKB, caused an increase in the intracellular calcium concentration as measured by the fluorescent dye Fura-2. The maximal change in intra-cellular calcium induced by 10 microM SP-(7-11) was 140 +/- 30 nM. We postulate that cleavage of SP by neutral endopeptidases which are present in the synovial fluid and which yield SP-(7-11) may be of biological importance in chondrocyte-mediated cartilage pathology.

Positively reinforcing effects of the neurokinin substance P in the basal forebrain: Mediation by its C-terminal sequence

The conditioned corral preference paradigm was used to assess reinforcing effects of substance P (SP) and its N- and C-terminal fragments injected unilaterally into the region of the nucleus basalis magnocellularis (NBM) in rats. Behavioral testing was carried out in a circular open field, consisting of 4 quadrants equally preferred by the animals prior to conditioning. A single conditioning trial was performed. Rats received one microinjection (0.5μl) of SP (0.74 pmol), of the N-terminal fragment SP(1–7) and the C-terminal fragment analog DiMe-C7 (each at doses of 0.074, 0.74, and 74 pmol), or vehicle (phosphate-buffered saline; PBS). After injection the rats were placed into the open field with the four quadrants being separated by Plexiglas barriers (closed corral). During the test for conditioned corral preference, when provided a choice between the four quadrants, only those rats injected with SP and the equimolar dose of DiMe-C7 (0.74 pmol) spent more time in the treatment corral, indicative of a positively reinforcing action. None of the other doses of DiMe-C7 and of SP(1–7) influenced the preference behavior. For rats injected with 0.74 pmol SP, SP(1–7), and DiMe-C7, a behavioral analysis was performed for the 15 min conditioning trial. SP and DiMe-C7 reduced rearing and grooming behavior, whereas DiMe-C7 and SP(1–7) increased locomotor activity. However, the acute behavioral effects of SP and its fragments were not correlated with the subsequent place preference behavior during the test trial. The results are discussed in the framework of a structure/activity relationship for the positively reinforcing properties of SP in the region of the NBM. Furthermore, neuropathological implications of the present data are considered, since the homologous nucleus basalis of Meynert in man is known to degenerate in Alzheimer's disease, which is characterized behaviorally by a progressive deterioration in associative functioning.

Studies on the vascular permeability induced by intrathecal substance P and bradykinin in the rat

The effects of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin (NKB) and selective agonists for neurokinin receptors were assessed on cutaneous vascular permeability after intrathecal (i.t.) administration in rats. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency ([p-Glu 6] SP-(6–11)>SP⩾SP-(4–11)>[p-Glu 5, MePhe 8, Sar 9]SP-(5–11)=[p-Glu 5]SP-(5–11)>SP-(7–11) an d SP>NKA>NKB). The N-terminal fragments SP-(1–4), SP-(1–7) and SP-(1–9) were inactive. The NK-1 receptor selective agonists ([β-Ala 4, ar 9, Met(O 2) 11]SP-(4–11) and [Pro 9,Met(O 2) 11]SP) were more potent than the NK-2 ([Nle 10]NKA-(4–10)) and NK-3 ([β-Asp 4,MePhe 7]NKB-(4–10) and [MePhe 7]NKB) receptor-selective agonists. Plasma extravasation was also increased by i.t. bradykinin (BK, 8.1 nmol) while the fragment BK-(1–8), a potent B 1-receptor-selective agonist, produced only a slight effect at 81 nmol. When BK was given after prior i.t. administration of 6.1 nmol of [Thi 5,8,D-Phe 7]BK, an antagonist of BK at the B 2-receptor, the increase in vascular permeability was significantly attenuated. The analogue [Leu 8]BK-(1–8) (10.3 nmol), an antagonist of BK at the B 1-receptor, failed to modify the BK-induced plasma extravasation. Plasma extravasation induced by SP (6.5 nmol) and BK (8.1 nmol) was abolished in cervically vagotomized rats, and significantly reduced in both spinal rats and in capsaicin-treated animals. Conversely, bilateral adrenalectomy (48 h earlier) and intercollicular decerebration (30 min earlier) had no major effect on the response elicited either by SP or BK. The response to SP remained unaffected by methysergide and hexamethonium but was significantly reduced by methylnitrate atropine and diphenhydramine. Indomethacin significantly enhanced the plasma extravasation induced by SP. These results suggest that SP and BK may play a role as spinal mediators in peripheral vascular permeability through a sensory and cholinergic vagal mechanism involving a spinobulbar pathway. The receptors mediating the response to SP and BK in the spinal cord are of the NK-1 and B 2 subtypes, respectively.

Substance P enhancement of inhibitory avoidance learning: Mediation by the N-terminal sequence

Experiments were performed to investigate the effects of intraperitoneally administered undecapeptide substance P (SP), its N-terminal fragment SP(1–7) (SPN) and the C-terminal analog [pGlu 6]-SP(6–11) (SPC) on inhibitory avoidance learning, using a one-trial up-hill avoidance task. In Experiment 1 rats were injected with either SP (50 μg/kg), SPN (3.3, 33, 167, 333 μg/kg) or SPC (2.7, 27, 134, 268 μg/kg) immediately after the training trial. Controls received the diluent vehicles. When tested 24 hr later, rats injected with 50 μg/kg SP (37 nmol/kg) and 167 μg/kg SPN (185 nmol/kg) exhibited longer step-up latencies than vehicle-treated controls. None of the other doses of SPN nor of the C-terminal fragment influenced performance. In Experiment 2, 167 μg/kg SPN or vehicle was injected posttrial either immediately or 5 hr after the training trial. Retention latencies 24 hr later were longer for rats treated with 167 μg/kg SPN immediately after the training trial. Performance of the SPN 5-hr delay group did not differ from that of the vehicle-injected controls, ruling out proactive effects of SPN on recall.

Inhibition of angiotensin converting enzyme by N-terminal fragments of substance P

A range of N-terminal fragments of substance P (SP) were evaluated for inhibitory activity against angiotensin converting enzyme (ACE) from rat lung and brain (striatum). SP inhibited the enzyme from both sources in a concentration dependent manner (IC 50 30 μM). The N-terminal fragments SP[1–7], SP[1–6], SP[1–4] and SP[3–4] were equipotent with SP for both sources of the enzyme. However, SP[1–3] showed a difference in its activity, being more active than SP (IC 50 10 μM) in inhibiting the brain enzyme, but inactive against lung ACE. These results suggest that the inhibitory action of SP on ACE resides in the N-terminus of the peptide. The difference in reactivity towards SP[1–3] lends support to the idea that lung and brain ACE are different isozymes.

Capillary permeability induced by intravenous neurokinins

The effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP greater than NKA greater than NKB) for neurokinins and (SP greater than [p-Glu6]SP(6-11) greater than SP(4-11) greater than [p-Glu5]SP(5-11) greater than SP(7-11] for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu5, MePhe8, Sar9]SP(5-11) was slightly more potent than [p-Glu5]SP(5-11). The N-terminal fragments SP(1-4), SP(1-7) and SP(1-9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar9, Met(O2)11]SP and [beta-Ala4, Sar9, Met (O2)11]SP(4-11) were more potent than the NK-2 [( Nle10]NKA(4-10] and NK-3 [( MePhe7]NKB and [beta-Asp4, MePhe7]NKB(4-10] receptor selective agonists. Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of D-Arg0[Hyp3.D-Phe7]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar9, Met(O2)11]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [beta-Ala4, Sar9, Met(O2)11]SP(4-11), SP(4-11) and [p-Glu6]SP(6-11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an increase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP. These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT (5-HT1 and 5-HT2 receptors), histamine (H1 receptors) and kinins (B2 receptors) in the response to SP and indicate that the two positively charged amino acids (Arg, Lys) at the N-terminal end of the SP molecule are essential to trigger the release of prostaglandins from mast cells. This mechanism is responsible for the indirect effect of SP and related peptides on capillary permeability and does not appear to be mediated by a selective SP receptor. In addition, neurokinins may increase capillary permeability by direct activation of a NK-1 receptor type on the vascular endothelium.