In vivo immunomodulation by the neuropeptide substance P.

Many experiments have demonstrated that the nervous and immune systems interact in a bidirectional fashion. Neuropeptides, including substance P, have been shown to modulate lymphocyte DNA, RNA and immunoglobulin synthesis in vitro and to play a role in inflammatory and hypersensitivity disease states. However, the role of substance P as an immunomodulator in vivo is uncertain and there is only indirect evidence of this effect obtained in vitro. Therefore, we have assessed the effect of substance in vivo on DNA and immunoglobulin synthesis by murine splenic and Peyer's patch lymphocytes after the continuous administration via a miniosmotic pump of substance P in vivo. Substance P administered in this fashion increased cell proliferation of lymphocytes isolated from both organs. Immunoglobulin synthesis was also increased and in a relatively isotype-specific manner. IgA synthesis was most affected, IgM synthesis less so and IgG synthesis was not changed significantly. These effects of substance P on lymphocytes in vivo are similar to its effects on cell proliferation and immunoglobulin synthesis when cells are exposed to this peptide in vitro. These results provide direct evidence that neuropeptides (substance P) may modulate lymphocyte function in vivo and that neuropeptides should be incorporated into the conceptual framework of immune regulation.     

Kappa-/mu-receptor interactions in the opioid control of the in vivo release of substance P-like material from the rat spinal cord.

The possible involvement of mu and kappa receptors in the opioid control of the spinal release of substance P-like material was assessed in vivo, in halothane-anaesthetized rats whose intrathecal space was continuously perfused with an artificial cerebrospinal fluid supplemented with various opioid receptor agonists and antagonists. Whereas the intrathecal perfusion with the mu agonist DAGO (10 microM) significantly enhanced (approximately + 50%) the spontaneous release of substance P-like material, that with the kappa agonist U 50488 H (10 microM) produced no change in the peptide outflow. The respective antagonists naloxone (10 microM) for the mu receptors and nor-binaltorphimine (10 microM) for the kappa receptors did not affect the spontaneous release of substance P-like material, indicating that endogenous opioids acting at mu and kappa receptors do not exert a tonic control on substance P-containing neurons in the spinal cord of halothane-anaesthetized rats. However, as expected from the involvement of mu receptors, the stimulatory effect of DAGO on the peptide outflow could be prevented by naloxone but not norbinaltorphimine. Furthermore, instead of an increase with DAGO alone, a significant decrease in the spinal release of substance P-like material was observed upon the intrathecal perfusion with DAGO plus U 50488 H. Additional experiments with the respective mu and kappa antagonists naloxone and nor-binaltorphimine demonstrated that this effect actually resulted from the simultaneous stimulation of mu and kappa receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological control of emotion-related behaviors by endogenous enkephalins involves essentially the delta opioid receptors

The endogenous pentapeptide enkephalins bind to the mu and delta opioid receptors, with a slightly higher affinity for the latter. It remains a controversy regarding the respective physiological role of mu and delta opioid receptors in the control of emotion and motivation. One of the difficulties to investigate this problem is the low tonic extracellular release of enkephalins in various brain structures. To overcome this problem the synaptic levels of these pentapeptides were enhanced by inhibition of enzymes involved in their catabolism with the selective inhibitor H3N-CH(CH2-CH2-S-CH3)-CH2-S-S-CH2-CH(CH2phi)-CONH-CH(CH2phi)-COOCH2phi (RB101). This compound was shown to increase the extracellular levels and lifetime of endogenous enkephalins. Similar responses were obtained in wild-type and mu opioid receptor knockout mice following RB 101 administration in behavioral tests measuring locomotor activity, anxiety (elevated O-maze), and motivation (forced swim test and conditioned suppression of motility). In contrast, RB 101 led to antinociceptive responses only in wild-type animals using hot plate and tail immersion tests. These results clearly demonstrate the critical role of delta opioid receptors activated by the endogenous opioid peptides, in the physiological control of emotion- and motivation-related behaviors. In contrast, antinociceptive modulation, at least with respect to thermal nociceptive stimuli, involves enkephalin-activated mu opioid receptors. These findings could open new perspectives in the treatment of mood disorders using either inhibitors of enkephalin catabolism or delta opioid agonists.     

Co-localization of N-methyl-D-aspartate receptors and substance P (neurokinin-1) receptors in rat spinal cord

Glutamate, substance P (SP), and their receptors have been implicated in the initiation and maintenance of persistent pain through an interaction at second order spinal cord neurons. Employing well-characterized antibodies to the SP receptor and the N-methyl-D-aspartate receptor (NR1 subunit, splice variant missing exon 22), we demonstrate co-localization of these receptors on second order neurons at cervical, thoracic, lumbar, and sacral spinal cord levels. The co-localization was marked in lamina I of the dorsal horn at all levels and in the intermediolateral nucleus of the thoraco-lumbar spinal cord nuclei associated with autonomic function.     

In vivo release of substance P in cat dorsal horn studied with microdialysis

Variations in the extracellular concentration of substance P (SP) were measured in cat dorsal horn in vivo by microdialysis and radioimmunoassay. Electrical unilateral stimulation of the sciatic nerve was used to evoke release of SP. At high-intensity stimulation, activating slowly conducting (approx. 0.9 m/s) fibres, there was an increase in substance P-like immunoreactivity (SP-LI) to 338% of the preceding control value (P less than 0.05, n = 5) in the dialysates collected at levels L6-L7 at the stimulated side. A less pronounced increase, to 164% of the basal level, was found at the contralateral side. More cranially (L1-L5) in the dorsal horn at the stimulated side, only a moderate or no increase in SP-LI was seen. Stimulation with low intensity, sufficient to activate fast (greater than 20 m/s), but not slowly conducting fibres, evoked no increase in SP-LI. The results indicate that unilateral peripheral C-fibre activation induces a local SP release in the dorsal horn present also on the side not stimulated.     

In vivo release of substance P in the nucleus tractus solitarii increases during hypoxia

In vivo release of substance P (SP) was measured by microdialysis in the nucleus tractus solitarii (nTS) in adult cats. Small perfused semipermeable tubules (microdialysis probes) were implanted stereotaxically in the nTS, at the position of respiration-related neurons and perfused with artificial CSF. SP was determined by radioimmunoassay of the perfusate. Increased extracellular concentrations of SP-like immunoreactivity (SP-LI) were measured during hypoxia induced in artificially ventilated cats. In addition, a prolonged increase in the extracellular SP-LI concentration was encountered after cervical vagotomy. The results corroborate the suggestion that SP is a mediator of the central response to hypoxia.     

Capsaicin induced release of multiple tachykinins (substance P, neurokinin A and eledoisin-like material) from guinea-pig spinal cord and ureter

The release of tachykinins from isolated slice preparations of the guinea-pig spinal cord and ureter was studied in vitro. Capsaicin (10 microM) caused release of substance P, neurokinin A and an eledoisin-like component from both the spinal cord and ureter. The release of tachykinins induced by capsaicin or potassium (60 mM) was calcium dependent. No detectable release of neurokinin B or neuropeptide K, an N-terminally extended form of neurokinin A, was induced by capsaicin. No detectable release of tachykinins could be demonstrated after exposure to agents which are known to activate C-fibre afferents, such as histamine, bradykinin, serotonin, prostaglandins E1, E2 or acetylcholine. Protein extravasation in the ureter, as determined by the Evans Blue extravasation technique was used as a functional correlate to the tachykinin release. Protein extravasation was induced in vivo by local intraluminal injections of capsaicin at several hundred-fold lower concentrations than those required to induce a detectable release of tachykinins in vitro. The difference may, however, partly depend on the experimental conditions and the detection limit of the tachykinin assay used. The protein extravasation response to capsaicin was absent after systemic capsaicin pretreatment, which causes a marked depletion of tachykinins in the ureter. In conclusion, capsaicin evokes release of several tachykinins from both central and peripheral endings of primary afferent neurons. The peptides released from sensory nerves in the periphery may induce effects such as protein extravasation and smooth muscle contraction.     

Differential inhibitory/stimulatory modulation of spinal CCK release by mu and delta opioid agonists, and selective blockade of mu-dependent inhibition by kappa receptor stimulation.

Opioid-cholecystokinin (CCK) interactions at the spinal level were investigated by looking for possible modulations by various opioid agonists of the release of cholecystokinin-like material (CCKLM) from slices of the dorsal zone of the rat lumbar enlargement. K(+)-evoked CCKLM overflow was reduced by 0.1-10 microM of the mu agonist DAGO or 10 nM to 3 microM of the delta agonist DTLET. By contrast, at a higer concentration (10 microM), the latter drug as well as morphine enhanced CCKLM overflow. Although inactive alone, the kappa opioid agonist U 50488 H (1 microM) prevented the inhibitory effect of DAGO without affecting that of DTLET. These data suggest that an opioid acting through the stimulation of mu, delta and kappa receptors (such as morphine) should produce a net increase in the spinal release of CCK.     

Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction

At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 muM) and the slowly hydrolysable ATP analog 5'-adenylylimidodiphosphate lithium (betagamma-imido ATP) (30 muM) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and betagamma-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A(1) adenosine receptor antagonist and alpha,beta-methylene ADP sodium salt (alphabeta-MeADP), which is an inhibitor of ecto-5'-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca(2+) channels by both, Cd(2+) or the combined application of nitrendipine and omega-conotoxin GVIA (omega-CgTx) (L-type and N-type Ca(2+) channel antagonists, respectively) prevented the effect of betagamma-imido ATP, indicating that the nucleotide modulates Ca(2+) influx through the voltage-dependent Ca(2+) channels related to spontaneous secretion. betagamma-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of G(i/o) proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors coupled to G(i/o) proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of betagamma-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. betagamma-Imido ATP did not affect 10, 15 and 20 mM K(+)-evoked release and application of reactive blue-2 before incubation in high K(+) induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca(2+) channels related to tonic secretion through the activation of P2Y receptors coupled to G(i/o) proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K(+), endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.     

NMDA receptors in primary afferents require phosphorylation by Src family kinases to induce substance P release in the rat spinal cord

The function of N-methyl-d-aspartate (NMDA) receptors in primary afferents remains controversial, in particular regarding their ability to evoke substance P release in the spinal cord. The objective of this study was, first, to confirm that substance P release evoked by NMDA is mediated by NMDA receptors in primary afferent terminals. Second, we investigated whether these NMDA receptors are inactivated in some conditions, which would explain why their effect on substance P release was not observed in some studies. Substance P release was induced in spinal cord slices and measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. NMDA (combined with d-serine) induced NK1 receptor internalization with a half of the effective concentration (EC₅₀) of 258 nM. NMDA-induced NK1 receptor internalization was abolished by the NK1 receptor antagonist L-703,606, confirming that is was caused by substance P release, by NMDA receptor antagonists (MK1801 and ifenprodil), showing that it was mediated by NMDA receptors containing the NR2B subunit, and by preincubating the slices with capsaicin, showing that the substance P release was from primary afferents. However, it was not affected by lidocaine and ω-conotoxin MVIIA, which block Na⁺ channels and voltage-dependent Ca²⁺ channels, respectively. Therefore, NMDA-induced substance P release does not require firing of primary afferents or the opening of Ca²⁺ channels, which is consistent with the idea that NMDA receptors induce substance P directly by letting Ca²⁺ into primary afferent terminals. Importantly, NMDA-induced substance P release was eliminated by preincubating the slices for 1 h with the Src family kinase inhibitors PP1 and dasatinib, and was substantially increased by the protein tyrosine phosphatase inhibitor BVT948. In contrast, PP1 did not affect NK1 receptor internalization induced by capsaicin. These results show that tyrosine-phosphorylation of these NMDA receptors is regulated by the opposite actions of Src family kinases and protein tyrosine phosphatases, and is required to induce substance P release.     

Modulation of somatocardiac sympathetic reflexes mediated by opioid receptors at the spinal and brainstem level

This study investigated astroglial responses after focal cerebral ischemia in the rat cortex induced by photothrombosis. Astrocyte activation was studied at various time points by immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin (VIM). We found a dual astrocytic response to focal ischemia: In the border zone of the infarct, GFAP-positive astrocytes were present within 2 days and persisted for 10 weeks. These astrocytes additionally expressed VIM. Remote from the ischemic lesion, cortical astrocytes of the entire ipsilateral hemisphere transiently expressed GFAP, but not VIM, beginning on day 3 after photothrombosis. This response had disappeared on day 14. By recording DC potentials, five to seven spreading depressions (SD) could be detected on the cortical surface during the first 2 h after photothrombosis. Treatment with MK801, a non-competitive NMDA-receptor antagonist, completely abolished SD and remote ipsilateral astrocytic activation, while the reaction in the border zone of the infarct remained unchanged. Functionally, persistent astrocytosis around the infarct might be induced by leukocyte-derived cytokines, while NMDA-receptor-mediated SD might cause remote responses.     

Presynaptic localization of substance P degradative enzymes(s) in rat substantia nigra.

Intrastriatal injections of kainic acid in rat brain, which destroyed striatal nerve cell bodies and their axons projecting to the substantia nigra (s. nigra) decreased the rate of breakdown of exogenuously added substance P by washed slice preparations of s. nigra. Injection of 6-hydroxydopamine (6-OHDA) into the s. nigra, which destroys the dopamine nerve cell bodies in this region, did not significantly affect the rate of degradation of substance P by nigral slices. Part of the peptidase activity responsible for breakdown of substance P in the s. nigra may thus be located on the terminals of striatal afferents to the s. nigra.     

GABAA and GABAB antagonists prevent the opioid inhibition of endogenous acetylcholine release evoked by glutamate from rat neostriatal slices

We have examined the role of the GABAergic system in the opioid inhibition of endogenous acetylcholine (ACh) release from rat neostriatal slices by blocking either γ-aminobutyric acid-A (GABA_A) or GABA_B receptors. GABAergic antagonists (bicuculline or phaclofen) completely blocked μ- (morphine or DAGO) and δ-opioid (DPDPE) inhibition of glutamate-evoked endogenous ACh release in a concentration-dependent manner. However, GABA antagonists were ineffective in blocking the opioid inhibition of potassium-evoked endogenous ACh release. These findings point to the important role of the GABAergic system in the regulation of μ- and δ-opioid inhibition of cholinergic neurons stimulated by glutamate.     

Direct inhibition of substantia gelatinosa neurones in the rat spinal cord by activation of dopamine D2-like receptors

Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. In current-clamp mode of patch-clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage-clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs⁺ or GDP-β-S in the pipette solution, and was inhibited in the presence of Ba⁺. The DA-induced current reversed its polarity at a potential close to the equilibrium potential of the K⁺ channel calculated from the Nernst equation. The DA-induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA-induced outward current was suppressed by a D2-like receptor antagonist, sulpiride, but not by a D1-like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G-protein-mediated activation of K⁺ channels through D2-like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.     

Neonatal capsaicin treatment abolishes the modulations by opioids of substance P release from rat spinal cord slices

The possible modulation by opioids of substance P (SP) release at the spinal level was studied using slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Capsaicin (0.5 microM) selectively evoked a Ca2+-dependent overflow of SP-like material (SPLI) from primary afferent fibers which was enhanced in the presence of mu-opioid agonists (DAGO, FK 33824, sufentanyl, morphine), reduced by the delta-opioid agonist DTLET, and unaltered by the kappa-opioid agonist U 50488 H. Selective antagonists (naloxone, ICI 154129) prevented the effects of mu- and delta-opioid agonists. Neonatal capsaicin (50 mg/kg) abolished the stimulatory effect of in vitro capsaicin (0.5 microM) but not that of 30 mM K+ on SPLI outflow. This K+-induced SPLI release was unaffected by opioids. Presynaptic inhibitory control of SPLI release from capsaicin-sensitive primary afferent fibers might account for the analgesic effect of delta- but not mu- and kappa-opioid agonists at the spinal level.     

Depletion of substance P from rat primary sensory neurons by ATP, an implication of P2X receptor-mediated release of substance P.

Effects of ATP on substance P immunoreactivity were examined in cultured dorsal root ganglion neurons. We found that treatment of dorsal root ganglion neurons with ATP significantly depleted substance P immunoreactivity on the neurites and somata of the neurons. The effects of ATP were significantly inhibited by the purinergic P2 receptor antagonists suramin (30 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (10 microM). We also showed that ATP-induced depletion of substance P immunoreactivity from dorsal root ganglion neurons depended on the entry of Ca(2+). In a spinal cord slice preparation, we also found the internalization of neurokinin-1/substance P receptors in many dorsal horn neurons following the application of ATP or alpha,beta-methylene-ATP.Together these results indicate that activation of P2X receptors may result in release of substance P from primary afferent neurons.     

Effects of peripheral nerve injury on delta opioid receptor (DOR) immunoreactivity in the rat spinal cord

Morphine and other opioids have direct analgesic actions in the spinal cord and chronic spinal administration of opioid agonists is used clinically in patients suffering from severe, chronic pain. Neuropathic pain resulting from peripheral nerve injury is often less sensitive to opioid therapy than other forms of chronic pain in both humans and animal models. Changes in spinal mu-opioid receptor (MOR) expression have been demonstrated in animal models of neuropathic pain. However, these changes alone fail to account for the attenuation of opioid activity. Reduced expression of delta-opioid receptors (DOR) following peripheral nerve injury has been reported but most of these reports are limited to subjective observation. The magnitude and consistency of these changes is therefore unclear. In addition, previous studies did not evaluate the effects of nerve injury on behavioral measures to confirm induction of aberrant pain symptoms. We therefore performed quantitative image analysis to evaluate the effect of peripheral nerve injury on DOR-immunoreactivity in spinal cord sections from rats previously characterized for sensory responsiveness. We observed statistically significant decreases ipsilateral to nerve injury in all three models tested: sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. These results suggest that decreases in the expression of DOR are a common feature of peripheral nerve injury.     

Is substance P released from slices of the rat spinal cord inactivated by peptidase(s) distinct from both 'enkephalinase' and 'angiotensin-converting enzyme'?

Studies on the effects of peptidase inhibitors on substance P-like immunoreactive material (SPLI) released by K(+)-induced depolarization from slices of the rat spinal cord showed that bacitracin was the most potent agent to protect SPLI from degradation. Captopril and thiorphan which inhibit, respectively, angiotensin I converting enzyme and endopeptidase-24.11 also protected SPLI from degradation. However other inhibitors of these two enzymes, kelatorphan for endopeptidase-24.11 and enalaprilat for angiotensin I converting enzyme were essentially inactive, indicating that both enzymes are probably not involved in the degradation of endogenous substance P. Instead, the non-additive protecting effect of bacitracin, captopril and thiorphan might be due to the blockade of some 'bacitracin-sensitive enzyme' playing a key role in the catabolism of SP within the rat spinal cord.     

长效抗生育埋植剂CaproF体内药物释放的研究

目的对可降解长效抗生育埋植剂CaproF的体内药物释放动力学进行评价。方法将CaproF植入Wister大鼠皮下。每隔一定时间处死动物,取出埋植剂,用紫外分光光度法测药物残留量,计算单位长度埋植剂平均每日药物释放量。放射免疫法测定左炔诺孕酮(LNG)血药浓度。结果CaproF埋植剂在60、120、180、360、720d药物平均释放速率分别为(11.0±3.0)、(11.7±3.7)、(8.0±1.2)、(7.3±0.4)、(9.3±0.9)μg/(d·cm),并可维持基本恒定的血药浓度。结论左炔诺孕酮CaproF埋植剂植入体内后,平均药物释放速率达到7.6μg/(d·cm),并可维持2年的基本稳定释放。