Substance P N-terminal fragment SP(1-7) attenuates chronic morphine tolerance and affects dynorphin B and nociceptin in rats

The N-terminal substance P fragment SP_1-7 is known to modulate hyperalgesia and opioid withdrawal in animal models. This study examined the effects of intraperitoneal (i.p.) injections of SP_1-7 on chronic morphine tolerance and on the levels of dynorphin B (DYN B) and nociceptin/orphanin FQ (N/OFQ) in various brain areas of male Sprague-Dawley rats. Morphine tolerance was induced by subcutaneous injections of the opioid (10 mg/kg) twice daily for 7 days. SP_1-7 injected i.p. (185 nmol/kg) 30 min prior to morphine reduced the development of morphine tolerance. Immunoreactive (ir) DYN B and N/OFQ peptide levels were measured in several areas of the central nervous system. Levels of ir DYN B in rats treated with SP_1-7 and morphine were decreased in the nucleus accumbens, substantia nigra and ventral tegmental area and increased in the frontal cortex. The ir N/OFQ levels were increased in the periaqueductal gray and decreased in the nucleus accumbens. Since the concentration profiles of the two peptides were altered by SP_1-7 in the areas that are implicated in the modulation of opioid tolerance and analgesia, it is suggested that DYN B and N/OFQ systems may be involved in the effects of SP_1-7 on opioid tolerance.     

P物质与受体nk-1在结肠癌中的表达及定位

目的:了解P物质及其受体神经激肤1(NK-1)在结肠癌中的表达及定位,探讨其在结肠癌发病及临床诊断的意义.方法:应用免疫组化方法检测正常结肠、结肠癌组织及癌旁组织中SP及NK-1的表达.采用jmtjfx10[1].31 统计学软件处理数据,所得数据进行Q检验,结果:①在结肠癌组织中P物质阳性着色于胞浆,呈巢状或弥漫分布.②NK-1受体主要着色于细胞浆内,呈巢状或弥漫分布;少数为于细胞膜.③p物质与其受体NK-1在正常结肠粘膜组织及癌旁组织也有表达,位于细胞浆内,形态多呈圆形、椭圆形或不规则形.④二者在绝大多数结肠组织中呈强阳性表达,显著高于正常结肠粘膜组织和癌旁组织(p<0.01).结论:p物质与其受体NK-1在结肠癌组织中高度表达,提示神经内分泌参与了结肠癌的发生有关,说明P物质及NK-1受体可能参与了结肠癌的发病过程.     

A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system

A comparison of anatomical distributions of substance P (SP) and substance P receptors in the rat central nervous system was performed. SP was localized by microdissection and radioimmunoassay and SP fibers and cell bodies by immunohistochemistry. Receptors for ¹²⁵I-Bolton Hunter labelled SP (¹²⁵I-BH-SP) were characterized pharmacologically by a slice binding technique in sections that contained primarily striatum. The receptor was saturable and had an equilibrium dissociation constant (K_D) of 0.30 nM and maximum number of binding sites (B_max) of 37.8 fmol/mg protein. Pharmacological characterization using C terminal fragments and naturally occurring analogues of SP reflected characteristics of the receptor which had been shown previously in bioassays and biochemical assays. Comparison of distribution of SP fibers and cell bodies and SP receptors indicated that there is no consistent relationship between the amount of SP receptor and density of SP fibers or cell bodies in a given region of the brain.     

The effect of substance P1-7 amide on nociceptive threshold in diabetic mice

We previously demonstrated that intrathecal treatment with substance P metabolite substance P_1-7 induced anti-hyperalgesia in diabetic mice. In the present study, we have used a synthetic analog of this peptide, the substance P_1-7 amide, showing higher binding affinitiy than the native heptapeptide, for studies of the tail-flick response in diabetic and non-diabetic mice. Intrathecal injection of substance P_1-7 amide produced prolongation of the tail-flick latency in both diabetic and non-diabetic mice, an effect that was more pronounced in diabetic mice than non-diabetic mice. Moreover, the observed antinociceptive potency of the substance P_1-7 amide was higher in both diabetic and non-diabetic mice in comparison with the native substance P_1-7. The antinociceptive effect of substance P_1-7 amide was reversed by naloxone but not by the selective opioid receptor antagonist β-funaltrexamine, naltrindole or nor-binaltorphimine, selective for the μ-, δ- or κ-opioid receptor, respectively. In addition, the antinociceptive effect induced by substance P_1-7 amide was partly reversed by the σ₁ receptor agonist (+)-pentazocine, suggesting a possible involvement of the σ₁ receptor for the action of this peptide. These results suggest that the actions of substance P_1-7 amide mimic the effects of the native substance P fragment but with higher potency and that the mechanisms for its action may involve the σ₁ receptor system.     

Anxiolytic-like effects of substance P fragment (SP(1-7)) in non-human primates (Callithrix penicillata)

The behavioral effects of the amino (N)-terminal fragment of substance P (SP(1-7)) on the marmoset (Callithrix penicillata) predator confrontation test of fear/anxiety were investigated. The test apparatus consisted of a figure-eight maze with three parallel arms interconnected at each extremity to a perpendicular arm. A taxidermized oncilla cat (Felis tigrina) was placed outside the maze facing one of its corners. Subjects were submitted to seven 30 min maze habituation trials (HTs), in the absence of the 'predator', and then to six 30 min treatment trials (TTs), in the presence of the 'predator', consisting of four doses of SP(1-7) (5, 50, 250 and 500 microg/kg; IP), saline and sham injection. SP(1-7) treatment reversed, in a dose-dependent way, the fear-induced avoidance behavior due to the predator's presence and increased the frequency of exploratory behaviors. Locomotor activity decreased during successive HTs, yet increased after all SP(1-7) treatments. These results indicate that systemic administration of SP(1-7) produces anxiolytic-like effects in marmosets tested in the predator confrontation model of fear/anxiety.     

Distribution of substance P (SP) and vasoactive intestinal peptide (VIP) in pseudocapsules of uterine fibroids

The authors examined the presence of Substance P (SP) and Vasoactive Intestinal Polypeptide (VIP) and their related fibers in the pseudocapsule of uterine fibroids (PUF) and in normal myometrium (NM) during myomectomies in 57 non-pregnant women. 4 samples were removed from the normal myometrium (NM) and from PUF. The samples were sent for histological and immune-fluorescent investigations. SP and VIP values were found non-significantly higher in PUF than in NM: SP values were 10.2 ± 0.1 conventional units (C.U.) in PUF at the fundus of the uterus (FU) vs. 8.1 ± 0.6 C.U. of NM in the FU (p > 0.05), and SP values were 25.1 ± 0.9 C.U. in PUF in the uterine body (UB) compared to. 23.2 ± 1.4 C.U. of NM in the myometrium of the UB (p > 0.05). VIP values were 11.5 ± 0.9 C.U. in the PUF in FU compared to 9.8 ± 1.4 C.U. of NM in the FU (p > 0.05), and VIP values were 33.9 ± 3.9 C.U. in the PUF in the UB vs. 32.6 ± 4.8 C.U. of the NM in the UB (p > 0.05). These findings show that SP and VIP neurofibers are present in the fibroid pseudocapsule, similar to the values in the normal myometrium of a non-pregnant uterus. An intracapsular myoma excision which respects the pseudocapsule permits a physiological healing process of the uterine scar, due to a neurotransmitter sparing at the hysterotomic site. In women planning pregnancy, the myomectomy should be preferably performed respecting the pseudocapsule in order to preserve the neurotransmission.     

Distinct binding sites for substance P and neurokinin A (substance K): co-transmitters in rat brain

The distribution of binding sites in rat brain for iodinated neurokinin A and iodinated substance P were compared using autoradiography. Distinct patterns of binding for the two iodinated tachykinins were noted. Binding sites for iodinated neurokinin A were noted in the olfactory bulb, cortex, supraoptic n., paraventricular n., certain amygdaloid n., hippocampus, medial habenula, interpeduncular n., n. of the tractus solitarius, and dorsal horn of the spinal cord. This pattern was in contrast to low levels of binding of iodinated substance P to the cortex, supraoptic n., paraventricular n., and the interpeduncular n., but substantial density of binding sites in numerous other regions.     

Improving anticancer activity and selectivity of camptothecin through conjugation with releasable substance P

Substance P, an 11-residue neuropeptide, can be rapidly internalized through specific interaction with the neurokinin-1 receptor. Therefore, we designed and synthesized the substance P targeted camptothecin (CPT) conjugates via a releasable disulfide carbonate linker. All the conjugates exhibited comparable or stronger cytotoxicity to cancer cells that highly over-express neurokinin-1 receptor than free CPT. More importantly, the selectivity of conjugates was significantly improved compared with CPT. Our results indicated that these conjugates can be promising candidates for new chemotherapeutic drugs. In addition, increasing CPT loading or attachment of CPT to the C-terminal hexapeptide of substance P are useful strategies to enhance the therapeutic efficacy of substance P targeted conjugates.     

长爪沙鼠肠道生长抑素和P 物质细胞密度的年龄变化

胃肠激素对调节小哺乳动物的消化功能具有重要作用。本文应用卵白素- 生物素- 过氧化物酶复合物(Avidin-biotin-peroxidase complex,ABC)免疫组织化学法,对冬季幼年、成年和老年长爪沙鼠肠道生长抑素(Somtostatin,SS)和P 物质(Substance P,SP)细胞进行了定位和比较。结果显示:不同年龄组两种内分泌细胞的形态学特征相似,呈圆形、椭圆形、梭形、锥形或不规则形。SS 细胞随年龄增加而分布范围缩小,主要分布于小肠和十二指肠,老年鼠的结肠和直肠无分布;盲肠段老年鼠高于幼年鼠和成年鼠,其余各段均无年龄差异。幼年鼠、成年鼠和老年鼠SP 细胞分别以结肠、盲肠和直肠密度为最高,成年鼠结肠和直肠无分布;肠道各段的密度均有年龄差异。这些结果表明,长爪沙鼠肠道SS 和SP 细胞的分布模式和发育特点有年龄差异,这可能与其生存环境的食物质量和两种内分泌细胞相互拮抗的消化生理功能有关。     

Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1-7 in the rat spinal cord

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

血管紧张素—（1—7）——肾素血管紧张素系统的新激素

长期以来,血管紧张素－（１－７）「Ａｎｇ－（１－７））」－直被认为是血管紧张素Ⅱ的无生物活性代谢产物。近年的研究证明Ａｎｇ－（１－７）在神经系统和心血管功能调节中起有作用,是血管紧张素系统中一种新的重要激素。     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X₇ antagonists. These compounds were assayed for activity at both the human and rat P2X₇ receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X₇ receptors. Compounds 12 and 38 displayed hP2X₇pIC₅₀s >7.8 with less than 2-fold difference in potency at the rP2X₇.     

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

Effects of bPTH fragments (1-34), (3-34) and (7-34) on uterine contraction

Synthetic bovine parathyroid hormone containing the NH2 terminal 34 amino acids [bPTH-(1-34)] was recently demonstrated to inhibit oxytocin stimulated uterine contraction in vitro. The parathyroid hormone analogues [Nle8, Nle18, Tyr34]bPTH-(3-34)amide [NTA-(3-34)] and [Tyr34]bPTH-(7-34)amide [NTA-(7-34)] have been reported to act as inhibitors of antagonists of parathyroid hormone (PTH) in numerous assays. In the present study the effects of these PTH analogues on uterine contraction and the ability of these analogues to act as antagonists to the uterine inhibitory action of bPTH-(1-34) in vitro were investigated. The NTA-(3-34) fragment had no effect on oxytocin stimulated uterine contractions. However, the NTA-(3-34) fragment was able to alter the ability of bPTH (1-34) to reduce oxytocin stimulated uterine contraction in a dose-related manner. Bovine PTH(1-34) (0.3 microgram/ml) reduced the contractile response obtained with oxytocin (0.5 mU/ml) by 20%. A dose of 15 micrograms/ml) of NTA-(3-34) abolished this inhibitory action of bPTH-(1-34) on oxytocin stimulated uterine contraction. In contrast the NTA-(7-34) caused a change in itself, stimulated contraction of resting uterine horns in a dose-related manner; 3.0 micrograms/ml of NTA-(7-34) caused a change in gram tension of + 1.5 grams. Bovine PTH-(1-34) was able to reduce the uterine contraction stimulated by NTA-(7-34) and 0.3 microgram/ml of bPTH-(1-34) reduced the contractile response obtained with 3.0 micrograms/ml of NTA-(7-34) by as much as 70%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Captopril intake decreases body weight gain via angiotensin-(1-7)

Angiotensin-(1-7) [Ang-(1-7)] plays a beneficial role in cardiovascular physiology by providing a counterbalance to the function of angiotensin II (Ang II). Although Ang II has been shown to be an adipokine secreted by adipocyte and affect lipid metabolism, the role of Ang-(1-7) in adipose tissue remains to be clarified. The aim of the present study was to investigate whether Ang-(1-7) affects lipid metabolism in adipose tissue. Ang-(1-7) increased glycerol release from primary adipocytes in a dose-dependent manner. A lipolytic effect of Ang-(1-7) was attenuated by pretreatment with A-779, a Mas receptor blocker and with an inhibitor of phosphoinositol 3-kinase (PI3K), or eNOS. However, losartan and PD123319 did not cause any change in Ang-(1-7)-induced lipolysis. Ang-(1-7)-induced lipolysis had an addictive effect with isoproterenol. In normal rats, chronic intake of captopril for 4 wks decreased body weight gain and the amount of adipose tissue and increased plasma Ang-(1-7) level. These effects were attenuated by administration of A-779. The levels of Mas receptor and phosphorylation of hormone-sensitive lipase (p-HSL) were significantly increased by treatment with captopril and these captopril-mediated effects were attenuated by the administration of A-779. There was no difference in diameter of adipocytes among sham, captopril- and captopril+A-779-treated groups. The similar effects of captopril on body weight, expression of Mas receptor, and p-HSL were observed in Ang-(1-7)-treated rats. These results suggest that captopril intake decreased body weight gain partly through Ang-(1-7)/Mas receptor/PI3K pathway.     

Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

P2X7 receptors regulate engulfing activity of non-stimulated resting astrocytes

We previously demonstrated that P2X7 receptors (P2X7Rs) expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but its roles in non-stimulated resting astrocytes remained unknown. It has been reported that astrocytes exhibit engulfing activity, and that the basal activity of P2X7Rs regulates the phagocytic activity of macrophages. In this study, therefore, we investigated whether P2X7Rs regulate the engulfing activity of mouse astrocytes. Uptake of non-opsonized beads by resting astrocytes derived from ddY-mouse cortex time-dependently increased, and the uptaken beads were detected in the intracellular space. The bead uptake was inhibited by cytochalasin D (CytD), an F-actin polymerization inhibitor, and agonists and antagonists of P2X7Rs apparently decreased the uptake. Spontaneous YO-PRO-1 uptake by ddY-mouse astrocytes was reduced by the agonists and antagonists of P2X7Rs, but not by CytD. Down-regulation of P2X7Rs using siRNA decreased the bead uptake by ddY-mouse astrocytes. In addition, compared to in the case of ddY-mouse astrocytes, SJL-mouse astrocytes exhibited higher YO-PRO-1 uptake activity, and their bead uptake was significantly greater. These findings suggest that resting astrocytes exhibit engulfing activity and that the activity is regulated, at least in part, by their P2X7Rs.