Inhibitory Effect Of Substance P Research Articles

Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus.

Disturbances in uterine contractile activity contribute to the development of inflammation, and recent evidence indicates that tachykinins, including substance P (SP) and neurokinin A (NKA), are involved in controlling uterine function. Here, we determined the effect of Escherichia coli (E. coli)-induced inflammation on expression of protein receptor subtypes for substance P (NK1R) and neurokinin A (NK2R) in the pig myometrium as well as their role in contractility of inflamed uterus. The severe acute endometritis developed in the E. coli group and the expression of NK1R and NK2R proteins increased in the myometrium. Compared to the pre-administration period, SP (10-6 M) reduced the amplitude and frequency in the myometrium of the E. coli group and the amplitude was higher and the frequency was lower versus other groups. NKA reduced the amplitude and increased the frequency in endometrium/myometrium of the E. coli group. In this group, the amplitude was lower and the frequency was higher than in the CON and SAL groups. Our research showed that NK2R (10-6 M) antagonist application abolished the NKA inhibitory effect on uterine amplitude. The application of the NK1R (10-5 M) antagonist together with SP revealed that the inhibitory effect of SP on uterine contractility is achieved independently of the NKR1. Additionally, taking into account the fact that NKA shows an inhibitory effect with the use of NK2R on uterine amplitude suggests the possibility of therapeutic use of the antagonist as a drug increasing uterine contractility in inflammation.

Response of porcine oocytes exposed to testosterone and substance P in vitro

The overproduction of androgens during the course of foliculogenesis may lead to disturbances in ovulation – the main reason for the establishment of the pathological state known as polycystic ovarian syndrome (PCOS). Furthermore, recently attention has been drawn to the involvement of tachykinins such as substance P (SP) in this process. Apart from this, both in animals and women, the above factors may impair oocyte development. Taking into account the aforementioned data we sought to find and compare the response of isolated porcine oocytes to different doses of testosterone (T) and SP with the goal of clarifying their effect on oocyte growth and meiosis progress. In our experimental approach porcine oocytes were exposed to these factors for 4h, 20h or 44h of culture. The obtained results reveal that both T and SP inhibit growth of porcine oocytes, cumulus cell expansion and meiotic resumption in vitro. Our data show that the inhibitory effect of SP on porcine oocytes is far weaker than the impact of T.

PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion

The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

An antinociceptive role for substance P in acid-induced chronic muscle pain

Release of substance P (SP) from nociceptive nerve fibers and activation of its receptor neurokinin 1 (NK1) are important effectors in the transmission of pain signals. Nonetheless, the role of SP in muscle pain remains unknown. Here we show that a single i.m. acid injection in mice lacking SP signaling by deletion of the tachykinin precursor 1 (Tac1) gene or coadministration of NK1 receptor antagonists produces long-lasting hyperalgesia rather than the transient hyperalgesia seen in control animals. The inhibitory effect of SP was found exclusively in neurons expressing acid-sensing ion channel 3, where SP enhances M-channel-like potassium currents through the NK1 receptor in a G protein-independent but tyrosine kinase-dependent manner. Furthermore, the SP signaling could alter action potential thresholds and modulate the expression of TTX-resistant sodium currents in medium-sized muscle nociceptors. Thus, i.m. SP mediates an unconventional NK1 receptor signal pathway to inhibit acid activation in muscle nociceptors, resulting in an unexpected antinociceptive effect against chronic mechanical hyperalgesia, here induced by repeated i.m. acid injection.

The inhibitory effect of Substance P on HCO3- secretion from the guinea pig microperfused pancreatic ducts

Background. We have previously shown using sealed, non-perfused, ducts that Substance P (SP) inhibits HCO3-secretion by inhibiting Cl-dependant HCO3-efflux on the luminal membrane of the duct cell (Hegyi et al. Am J Physiol Cell Physiol 285: C268-C276, 2003). However, in that study we had no direct evidence that SP affected a luminal anion exchanger. To address this issue we examined the effects of luminal H2DIDS on the inhibitory effect of SP in microperfused ducts.

Substance P inhibits pancreatic ductal HCO3-secretion via activation of protein kinase C in guinea pig

Background. The inhibitory control of pancreatic ductal HCO3- secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO3- secretion by modulating a Cl–dependent HCO3-efflux step at the apical membrane of the duct cell (Hegyi et al. Am J Physiol Cell Physiol 285: C268-C276, 2003). In the present study we have sought to localise SP in the guinea-pig pancreas and to identify the intracellular signalling pathway utilised by SP. Methods. Small intra/interlobular pancreatic ducts were isolated from guinea pigs. The rate of HCO3-secretion was determined from intracellular pH measurements as previously described (Hegyi et al., 2003). Western blot analysis was used to identify PKC isoforms expressed in the isolated ducts and immunohistochemistry to localize SP in the pancreas. Results. Our results show that SP is present in periductal nerves within the guinea-pig pancreas and that protein kinase C (PKC) mediates the effect of SP. Secretin (10 nM) stimulated HCO3- secretion by sealed, non-perfused, ducts about 3 fold and this effect was totally inhibited by SP (20 nM). Phorbol 12, 13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO3- secretion by about 40%, and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1µM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO3- secretion. Western blot analysis revealed that guinea pig pancreatic ducts express the alfa, -betaI, -delta, -epsilon, -eta, -theta, -zeta and -mu isoforms of PKC. Conclusions. Taken together, our results indicate that SP reduces HCO3- secretion in guinea pig ducts via activation of PKC, which inhibits a luminal Cl-/HCO3- exchanger (most likely a member of the SLC26 family). This work was supported by The Wellcome Trust and OTKA.

<b>Substance P induced enhancement of inhibitory synaptic </b><b>transmission in deep dorsal horn </b>

Substance P (SP) is the most popular neuropeptide currently considered in detail, and has been widely studied as an excitatory factor of nociception. However, analgesic effects were not clearly shown by inactivation of SP receptors by the use of SP receptor antagonists or SP knock-out mice in some recent studies. In order to investigate the mechanism, we studied the excitatory and inhibitory effects of SP and SP receptor agonists on deep dorsal horn (DH) neurons of the spinal slice preparation by using patch clamp method. We used neurokinin 1 (NK1) receptor agonist ([Sar9, Met(O2)11]SP: 1.0 µM), because SP has highest affinity for NK1 receptor in three SP receptors (NK1, NK2, and NK3). Bath application of SP or NK1 receptor agonist for 1 minute increased the frequency of spontaneous excitatory postsynaptic currents (sEPSC) mediated by glutamate in deep DH. SP and NK1 receptor agonist induced enhancement of glutamate release from interneurons and/or primary afferent fibers onto deep DH neurons. On the other hand, the application of SP or NK1 receptor agonist largely increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSC) and miniature inhibitory post synaptic currents (mIPSC) mediated by GABA and/or glycine in deep DH. These findings suggested that SP and NK1 receptors are expressed in GABAergic and/or glycinergic interneurons and that their activations facilitated GABA and/or glycine release onto deep DH neurons. In summary, it has been thought SP is generally participating in the enhancement of excitatory nociceptive pathway, but these results made clear that SP induced enhancement of inhibitory nociceptive transmission in deep DH. Clinical treatment of SP receptor antagonists against pain has been proposed for a long time, but is not established yet. Our observation in this study revealed it is necessary to consider the inhibitory effects of SP in the spinal deep dorsal horn in clinical application of SP receptor antagonists for the treatment of various pain sensation.

Substance P inhibits bicarbonate secretion from guinea pig pancreatic ducts by modulating an anion exchanger.

The stimulatory pathways controlling HCO3- secretion by the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, which may play an important role in the physiological control of the pancreas. The aim of this study was to investigate the cellular mechanism by which substance P (SP) inhibits pancreatic ductal HCO3- secretion. Small intra/interlobular ducts were isolated from the pancreas of guinea pigs. During overnight culture the ducts seal to form a closed sac. Transmembrane HCO3- fluxes were calculated from changes in intracellular pH (measured using the pH-sensitive dye BCECF) and the buffering capacity of the cells. We found that secretin can stimulate HCO3- secretion in guinea pig pancreatic ducts about fivefold and that this effect could be totally blocked by SP. The inhibitory effect of SP was relieved by spantide, an SP receptor antagonist. SP had no effect on the activity of basolateral Na+-HCO3- cotransporters and Na+/H+ exchangers. However, the peptide did inhibit a Cl--dependent HCO3- efflux (secretory) mechanism, most probably the Cl-/HCO3 exchanger on the apical membrane of the duct cell.

Substance P Abolishes the Facilitatory Effect of ATP on Spontaneous Glycine Release in Neurons of the Trigeminal Nucleus Pars Caudalis

Glycine release was facilitated by the activation of presynaptic ATP receptors (P(2X)-type) in a preparation of dissociated trigeminal nucleus pars caudalis neurons in which the native synaptic boutons were preserved. The action of ATP was completely blocked by substance P (SP) without alteration of the miniature IPSC (mIPSC) amplitude distribution. SP itself had no effect on mIPSC frequency or amplitude. The inhibitory effect of SP on ATP action was blocked by CP99994, indicating that the SP receptors are of the neurokinin-1 type. The ATP-induced facilitation of the mIPSC frequency was unaffected by Cd(2+). Moreover, SP did not inhibit the increase in mIPSC frequency induced high K(+) application, suggesting that SP did not modulate voltage-dependent calcium channels or subsequent steps in the release process. KT5720 and phorbol 12-myristate 13-acetate did not block SP action, indicating that neither the cAMP-protein kinase A nor the protein kinase C pathway mediates the SP effects. However, in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), SP was no longer able to inhibit the ATP-induced stimulation of mIPSC frequency. 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine also suppressed the SP action, suggesting that SP modulates P(2X) receptors via a Ca(2+)/calmodulin-dependent protein kinase II-mediated pathway. In conventional whole-cell mode, the presence of W-7 in the patch pipette did not affect the SP inhibitory action. Thus, SP is not likely to be generating its modulation through the production of a retrograde signal (involving calmodulin) from the postsynaptic cell to the presynaptic boutons. These results are the first demonstration of the modulation of one presynaptic receptor by another. Because SP inhibits the ATP stimulation of glycine release, SP may play a significant role in hyperalgesia or chronic pain.

Protein kinase-mediated reciprocal modulatory changes in anesthetic sensitivity of (BK)-K +- and GABA-A receptor-gated conductances in guinea-pig sympathetic neurons

(1) The interaction of substance P (SP)-mediated synaptic transmission with general anesthetics remains unknown. (2) Intracellular recordings were obtained from guinea-pig inferior mesenteric ganglion neurons to study monosynaptic responses to exogenous SP and GABA. (3) Propofol (1–100 μM) caused an increase in SP-evoked inward current responses and a concurrent decrease in peak amplitude of the afterspike hyperpolarization of intermittently evoked action potentials. These effects were occluded by the (BK)-K +-channel-selective blocker charybdotoxin (10 nM), and prevented by the protein kinase inhibitor staurosporine (100 nM). (4) Propofol also increased GABA-evoked current ( I GABA) responses. (5) When elicited during a SP response, I GABA was significantly diminished compared to control. In the presence of staurosporine (100 nM), the inhibitory effect of SP upon I GABA was abolished, and the propofol-induced augmentation of I GABA was significantly increased. (6) Thus, SP-evoked protein kinase activity produced reciprocal changes in anesthetic sensitivity of (BK)-K +- and GABA A-receptor-gated currents of these sympathetic neurons.

Ameliorative effects of tachykinins on scopolamine-induced impairment of spontaneous alternation performance in mice.

The present study was designed to clarify whether opioid neuronal systems are involved in the beneficial effects of tachykinins such as the neurokinin NK1 receptor agonist, substance P (SP), the neurokinin NK2 receptor agonist, neurokinin A (NKA), and the neurokinin NK3 receptor agonist, senktide, on the scopolamine-induced impairment of spontaneous alternation performance in mice. Intracerebroventricular injections of SP (0.1 microgram), NKA (0.3 microgram) and senktide (3 ng) inhibited the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance without influencing total arm entries, indicating the antiamnesic effects of tachykinins. Furthermore, the inhibitory effects of SP, but not those of NKA or senktide, were almost completely reversed by pretreatment with naloxone (1 mg/kg). However, the effects of SP on the scopolamine-induced impairment of spontaneous alternation performance were not influenced by pretreatment with the mu-opioid receptor antagonist, beta-funaltrexamine (5 micrograms), the delta-opioid receptor antagonist, naltrindole (4 ng), and the kappa-opioid receptor antagonist, nor-binaltorphimine (4 micrograms). These findings suggest that the effects of SP, unlike those of NKA or senktide, on the scopolamine-induced impairment of spontaneous alternation performance associated with spatial working memory are not mediated simply via a single type of opioid receptors, such as mu, delta or kappa.

Opposing gastric motor responses to TRH and substance P on their microinjection into nucleus raphe obscurus of rats.

Little is known about the functional role of putative neurotransmitters in the nucleus raphe obscurus (NRO) in the control of gastric motor function, although thyrotropin-releasing hormone (TRH) and substance P (SP) have been detected in the cell bodies and/or fibers of this nucleus. Therefore, we investigated the effects of microinjection of these peptides (in a volume of 60 nl) into the caudal NRO of alpha-chloralose-anesthetized rats while recording intragastric pressure, pyloric and greater curvature motility, and blood pressure. L-Glutamate (30 nmol) was first microinjected into the NRO to identify the "gastric" region of the NRO and elicited significant increases in intragastric pressure as well as pyloric and greater curvature motility in all 16 animals. TRH (2-45 pmol, n = 16) microinjected into the same sites increased intragastric pressure as well as pyloric and greater curvature motility, and these effects were abolished by bilateral cervical vagotomy and atropine (0.5-1.0 mg/kg iv) but not by spinal cord transection. Microinjection of SP (45-405 pmol, n = 15) into the same sites decreased intragastric pressure; however, the inhibitory effect of SP on pyloric and greater curvature motility did not attain statistical significance. The effect of SP on intragastric pressure was completely abolished by bilateral vagotomy but not by systemic administration of atropine (1 mg/kg) or spinal cord transection. Microinjections of 45 pmol TRH and 405 pmol SP just outside of the NRO did not result in changes in gastric function. No overall significant changes in blood pressure were noted after microinjection of L-glutamate, TRH, or SP into the gastric region of the NRO. We conclude that both TRH and SP affect gastric motor function in the caudal NRO via a vagally mediated pathway; TRH apparently activates vagal cholinergic pathways, but the mechanism of SP-evoked gastric motor inhibition remains to be further investigated.

Effects of Substance-P and Neuropeptide-Y onin VitroSteroid Release by Porcine Granulosa and Luteal Cells*

The presence of substance-P (SP)- and neuropeptide-Y (NPY)-like immunoreactivity was recently shown in nerves that innervate the ovary. In the present in vitro study we demonstrate that both peptides have direct effects on ovarian steroidogenesis. In cultured porcine granulosa (G-) cells, neither peptide affected progesterone (P) production under basal conditions, but they both inhibited gonadotropin-stimulated P secretion. In luteal (L-) cell cultures, basal as well as hCG-stimulated P release were dose-dependently inhibited by NPY (ED50, 4 x 10(-9) M; identical for both, basal and stimulated release), while SP had only a moderate inhibitory effect (ED50, 6 x 10(-8) M). In the presence of AP13, a specific SP antagonist, the inhibitory effect of SP on P release was abolished, which suggests a receptor-mediated effect. In addition, we determined androstenedione (A) and estradiol (E2) release into G- and L-cell culture media. While E2 production in G-cell cultures was not influenced by SP and NPY, both peptides had a dose-dependent stimulatory effect on E2 secretion by L-cells. In contrast to E2 release, A secretion by G- as well as L-cell cultures was increased by gonadotropins. Both SP and NPY decreased gonadotropin-stimulated A secretion by G- and L-cells under basal as well as hCG-stimulated conditions. Furthermore, we demonstrate SP immunoreactivity in media of G- and L-cell cultures with a HPLC retention time identical to that of synthetic SP. This may suggest ovarian synthesis, in which case the peptide exerts auto- and/or paracrine effects on ovarian steroidogenesis. From these in vitro results we suggest that SP and NPY have a modulatory effect on ovarian function in pigs not only by their well known regulatory effects of blood supply, but also by a direct effect on ovarian steroidogenesis.

Response of splanchnic-driven neurons to substance P and eledoisin-related peptide

The effects of iontophoretically applied substance P (SP) and eledoisin-related peptide (ERP) on the extracellularly recorded activity of spontaneous or d, l-homocysteic acid (DLH)-driven thoracic (T7–T8) spinal neurons, identified by orthodromic activation by the greater splanchnic nerve, were examined in chloralase/urethane anesthetized cats. Of 24 neurons tested, SP and/or ERP (18–120 nA) produced a weak (ca 30%) excitatory effect on 5 neurons, a weak inhibitory effect on 9 neurons, and no effect on the remaining neurons. When compared to the action of either DLH or GABA, we found both the excitatory and inhibitory effects of SP and/or ERP were delayed and prolonged. These data do not support a role for SP as a major neurotransmitter in spinal circuits in which visceral (splanchnic) afferents participate.

Effects of substance P, angiotensin II, oxotremorine and prostaglandin D2 on thyrotropin secretion in rats.

The effects of substance P (SP), angiotensin II, oxotremorine and prostaglandin D2 (PG D2) on thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) secretion in rats were studied. Either SP (100 micrograms/kg), angiotensin II (500 micrograms/kg), oxotremorine (1.0 mg/kg) or PGD2 (500 micrograms/kg) was injected intravenously or intraperitoneally, and the rats were serially decapitated. TRH, TSH and thyroid hormone were measured by means of a specific radioimmunoassay for each. The hypothalamic immunoreactive TRH (ir-TRH) contents were significantly increased by oxotremorine or SP and significantly decreased by angiotensin II, but no by PG D2. The plasma ir-TRH concentrations were significantly increased by angiotensin II, but not by oxotremorine, SP or PG D2. The plasma TSH levels were significantly increased by angiotensin II and significantly decreased by oxotremorine, SP or PG D2 in a dose-related manner. The plasma ir-TRH and TSH responses to cold were inhibited by oxotremorine, SP or PG D2, but enhanced by angiotensin II. The plasma TSH response to TRH was inhibited by SP, but enhanced by angiotensin II. The plasma TSH response to TRH did not differ from that of the control after PG D2 injection. In the haloperidol- or para-chlorophenylalanine (PCPA)-pretreated group, the inhibitory effect of PG D2 or oxotremorine on TSH release was prevented, while in the L-DOPA- or 5-hydroxytryptophan (5-HTP)-pretreated group, the inhibitory effect of SP on TSH release was prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of substance P and its C-terminal hexapeptide on gastric and pancreatic secretion in the dog.

In five dogs with gastric fistulas, Heidenhain pouches, and pancreatic fistulas, the effects of substance P (SP) and its C-terminal hexapeptide (SP6-11) on gastric acid and pancreatic secretions were determined under basal conditions and in response to secretory stimulation. SP or SP6-11 infused alone in graded doses (0.25-2.0 nmol.kg-1.h-1) caused a slight but significant increase in pancreatic secretions in fasted dogs, but, when given during the secretory stimulation, they caused significant inhibition of these secretions. They reduced gastric acid response to pentagastrin and peptone meal without affecting the serum gastrin level. They caused dose-dependent inhibition of secretin-induced pancreatic bicarbonate secretion and suppressed the pancreatic protein response to caerulein, feeding, and duodenal acidification. SP6-11 was equipotent on a molar basis with SP in the inhibition of gastric or pancreatic secretion, indicating that the C-terminal portion of SP exhibits a full spectrum of the biological action of the intact molecule. The inhibitory effects of SP and SP6-11 on the stomach and pancreas were observed at a dose range that was without any significant influence on the blood pressure, indicating that they are not caused by the interference of the blood flow to the pancreas.