鲑鱼降钙素(sCT)类似物在水溶液中的化学稳定性

用反相高效液相色谱和快原子轰击质谱方法(FAB-MS),研究了3个鲑鱼降钙素(sCT)类似物[Val¹,Ala⁷]sCT,[Val¹,Ala⁷,Ala³⁰]sCT和[D-Ala³⁰]sCT在不同pH和温度下水溶液中的化学稳定性,并对sCT及其类似物在pH9.0水溶液中的部分降解产物通过FAB-MS进行了确证。结果表明,sCT及其3个类似物在水溶液中的化学降解对pH依赖性较大,在碱性条件下易降解。提示不含二硫键的直链肽类似物[Val¹,Ala7]sCT和[Val1,Ala7,Ala30]sCT的化学稳定性比sCT高,而[D-Ala³⁰]sCT的化学稳定性则相对降低。     

促性腺激素释放多肽(GRP)对体外培养小鼠垂体分泌LH的影响

合成的促性腺激素释放多肽(GRP)及其类似物GRp～NH₂,[Glu^7.9.14Lys^6.10]GRP(6～14),[phe¹⁴]GRP(5～14)和[phe¹⁴]GRP浓度在0.05mmol·L^-1时,具有刺激体外培养的小鼠垂体分泌LH的作用。其活性依此相当对照垂体的115.4,114.2,140,160和179%。小鼠于妊振第7～9天或第1～5天,每只sc[phe¹⁴]GRP1mg·d^-1,或于妊娠第2～4天每只sc[phe¹⁴]GRP(5～14)1mg·d^-1,有40～60%的妊娠动物出现死胎。     

Different tachykinin receptors mediate chloride secretion in the distal colon through activation of submucosal neurones

We investigated the role of tachykinin receptor subtypes on secretory responses in the guinea-pig distal colon using Ussing chamber experiments and intracellular recordings from submucosal neurones. Choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) were demonstrated in submucosal neurones by immunohistochemistry. In Ussing chamber experiments substance P (SP), the NK-1-receptor agonist [SAR⁹,Met(O₂)¹¹]-SP and the NK-3-receptor agonist (MePhe⁷)-NKB increased dose-dependently short-circuit currents. The NK-2-receptor agonist (βAla⁸)-NKA(4–10) had no effect. Responses to 1–100 nM SP, [(SAR⁹,Met(O₂)¹¹]-SP and (MePhe⁷)-NKB were tetrodotoxin-sensitive but hexamethonium-insensitive. While (MePhe⁷)-NKB-responses were atropine-sensitive at all concentrations, the atropine sensitivity of the secretory responses to SP and [SAR⁹,Met(O₂)¹¹]-SP dramatically decreased with increasing concentrations. [SAR⁹,Met(O₂)¹¹]-SP and (MePhe⁷)-NKB effects were blocked by the selective NK-1 and NK-3 antagonists CP-99,994–1 (1 μM) and SR 142801 (1 μM), respectively. Combination of both antagonists blocked the SP-response. SR 142801 also suppressed the response to [SAR⁹,Met(O₂)¹¹]-SP. Desensitization with [SAR⁹,Met(O₂)¹¹]-SP significantly decreased (MePhe⁷)-NKB-responses but not vice versa. In intracellular recordings 90% of submucosal neurones were activated by both [SAR⁹,Met(O₂)¹¹]-SP and (MePhe⁷)-NKB as indicated by membrane depolarisation and enhanced spike discharge. These effects were tetrodotoxin-resistant and potentiated by atropine. NK-1- and NK-3-mediated responses occurred equally in ChAT-positive and in VIP-positive neurones. The results suggest the importance of NK-1- and NK-3-receptors on cholinergic and non-cholinergic submucosal neurones for secretory processes in the guinea-pig distal colon. Received: 16 June 1998 / Accepted: 22 September 1998     

孤啡肽及其片段的合成、痛觉调节作用和对免疫功能的影响

目的：研究孤啡肽(NC)与其4个片段(NC(1-15)NH₂, NC(1-13)NH₂, NC(1-11)NH₂, NC(1-5)NH₂) 在痛觉调节和免疫活性上的变化,探讨NC的构效关系。方法：固相多肽合成法合成NC及其片段;甩尾法测定它们对小鼠的痛敏作用和对吗啡镇痛作用的拮抗;T细胞玫瑰花结形成百分率和红细胞免疫粘附能力评测对免疫功能的影响。结果：虽然NC及其片段均有痛敏作用并可拮抗吗啡的镇痛作用,但NC(1-11)NH₂和NC(1-5)NH₂比母体活性约降低100倍,而NC(1-13)NH₂和NC(1-15)NH₂与母体有相同的活性。NC及片段(0.3～3 nmol.kg^-1)对T细胞免疫功能均有促进作用;NC(1-11)NH₂(0.3 nmol.kg^-1)对红细胞的免疫粘附能力有促进作用;NC(1-5)NH₂(0.3～30 nmol.kg^-1)不影响红细胞的免疫功能。结论：C端在NC的构效关系中有重要的作用。     

新皮啡肽I(DELI)类似物的合成及构效关系研究

用固相多肽合成法合成了类阿片肽新皮啡肽I(DELI)及其三个类似物(将Asp4从5位到7位逐一后移)。实验发现DELI在离体条件(浓度范围10^-14～10mol·L^-1)和在体条件(剂量范围0.5～5μg·kg^-1)下能提高红细胞玫瑰花环形成细胞百分率(E-RFC)和红细胞C3b受体花环百分率(RBC-CR₁),且可被纳洛酮阻断。这些结果表明DELI能提高大鼠免疫功能。实验还发现DELI及其类似物的镇痛和免疫活性次序(由大到小)分别是Asp⁴,Asp⁷,Asp⁵,Asp⁶和Asp⁴,Asp⁷,Asp⁶,Asp⁵。     

刺梨乙醇提取物体外抗氧化活性和α-葡萄糖苷酶抑制活性的研究

目的 研究刺梨体外抗氧化活性和α-葡萄糖苷酶抑制活性。方法 采用DPPH和ABTS氧化自由基清除试验对刺梨果肉乙醇提取物的抗氧化活性进行研究,并对其α-葡萄糖苷酶抑制活性进行了测定。结果 刺梨果肉乙醇提取物具有较强的DPPH氧化自由基清除能力[IC₅₀=(10.24±0.98)μg·mL^-1]和ABTS氧化自由基清除能力[IC₅₀=(4.92±0.31)μg·mL^-1]及α-葡萄糖苷酶抑制活性[IC₅₀=(5.75±0.71)μg·mL^-1]。结论 刺梨具有较好的抗氧化活性和α-葡萄糖苷酶抑制活性。     

有机锡化合物抗肿瘤生物活性研究

有机锡(IV)化合物能明显地抑制大鼠脑组织中PKC活性和肿瘤细胞增殖，且两者之间存在着相关性。抗肿瘤活性的构效关系是：(1)有机基团R决定整个化合物的生物活性，Ph>Et>n-Bu；(2)卤素的电负性影响化合物活性的大小。抗肿瘤活性可能通过[snR₂]²⁺实现。并能部分地阻断HL-60细胞周期中的G_I期向S期的移行。[SnPh₂F₂]，[SnPh₂(CysOS)]H₂O和[SnPh₂Cl2·phen(CH₃)₂]对PKC的IC₅₀值分别为25，15和20 umol·L^-1，对HL-60细胞的IC₅₀值分别为0．5，4．0和0．3umol·L^-1。但它们无诱导分化HL-60和K562细胞的作用。     

小檗碱对培养大鼠神经细胞内游离Ca2+的影响

以Fura-2/AM为细胞内钙离子的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了体外培养的新生大鼠神经细胞内游离钙([Ca²⁺]i)值,并观察了小檗碱(Ber)的影响。结果表明,Ber对神经细胞静息[Ca²⁺]i无明显影响,Ber1～100μmol·L^-1能剂量依赖地抑制去甲肾上腺素和H₂O₂引起的[Ca²⁺]i升高,其IC₅₀分别为39.9和17.9μmol·L^-1。高剂量Ber(10～100μmol·L^-1)能抑制高K+引起的[Ca²⁺]i升高。姐果提示,Ber对去甲肾上腺素,高K⁺及H₂O₂引起的[Ca²⁺]i升高的抑制作用可能是其抗脑缺血作用机制之一。     

前胡丙素对培养大鼠心肌细胞内游离Ca2+的影响

用Fura-2/AM技术直接观察前胡丙素(Pra-C)对培养大鼠心室肌细胞内游离钙的影响。结果显示Pra-C浓度为0.1～1.0μmol·L^-1可明显抑制CaCl₂,高K⁺和Bay K 8644引起[Ca²⁺]i增加,并且有剂量—效应关系,对ouabain引起的[Ca²⁺]i增加无明显作用。结果提示Pra-C降低心肌细胞[Ca²⁺]i的作用与抑制电压依赖性钙通道有关。     

3-甲基芬太尼立体异构体的合成、绝对构型和镇痛活性

报道3-甲基芬太尼(2)四个光学异构体的合成及其绝对构型的确定,并测定了各异构体的镇痛活性(小鼠,ip,热板法)。结果表明,cis-(+)-(3R,4S)-2的镇痛作用最强,ED₅₀0.00767 mg/kg,镇痛效能是吗啡的2600倍,比其对映体cis-(-)-(3S,4R)-2以及混旋的cis-(±)-2分别强119和1.5倍;trans-(+)-(3S,4S)-2的镇痛效能是吗啡的450倍、trans-(-)-(3R,4R)-2的4倍。     

强啡肽A－（1－13）类似物的合成及生物活性

用固相多肽合成方法，合成了强啡肽Ａ－（１－１３）（Ｉ）及其类似物［Ａｌａ８，Ｄ－Ｐｒｏ１０］－ＤＹＮＡ－（１－１３）－ＮＨ２（ＩＩ）和［Ｄ－Ａｌａ２，Ａｌａ８，Ｄ－Ｐｒｏ１０］－ＤＹＮＡ－（１－１３）－ＮＨ２（ＩＩＩ），并对其进行了镇痛活性试验和ＭＶＤ及ＲＶＤ试验。结果表明，合成产物均有镇痛活性，其中类似物ＩＩＩ的镇痛活性是１的３．６倍，ＲＶＤ试验活性比１强１３５倍，比已知的ｋ－受体强激动剂Ｕ５００４８８强１１倍。     

Synthesis and structure-activity relationships of dynorphin A-(1-8) amide analogues

In order to study the structure-activity relationships of dynorphin A-(1-8) amide [Dyn(1-8)-NH2], 20 analogues were synthesized by the solution method. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after intravenous administration. Some analogues that showed interesting activity in the bioassays and/or in the analgesic tests were further characterized in mu-, delta-, and kappa-representative binding assays. The obtained data indicate that modification of the enkephalin segment to give metabolically stable analogues with high affinity and selectivity for the kappa receptor is strictly limited and that introduction of MeArg in position 7 protects the Arg6-Arg-7 bond from enzymatic degradation without potency drop and change of opioid receptor selectivity. [MeTyr1,MeArg7,D-Leu8]Dyn(1-8)-NHEt (18) [IC50 (nM) = 0.3 (GPI), 7.4 (MVD), and 2.6 (RVD); tail pinch ED50 (mg/kg) = 0.75] showed opioid activity similar to that of dynorphin A in the three bioassays and relatively high kappa-receptor selectivity in the binding assays and produced a 2.5-fold more potent analgesic effect than morphine. [D-Cys2-Cys5,MeArg7,D-Leu8]Dyn(1-8)-NHEt (20) showed a 40-60-fold more potent opioid activity than 18 in the three bioassays and produced a 3.4-fold more potent analgesic effect than 18. In the binding assays, however, 20 showed higher affinity for mu and delta receptors than for the kappa receptor.     

β-Endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic β-endorphin (β-EP) analogs containing dermorphin or dynorphin-A-(1 – 13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH₂-terminal 1–7 segment of camel β-EP [β_c-EP-(1–7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1–7)] -β_c-EP was 120 times more potent than β_c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH₂-terminal 1–13 segment of human β-EP [β_h-EP-(1–13)] with dynorphin-A-(1–13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1–13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that β_c-EP-(8–31) facilitates the dermorphin moiety to act on opiate μ and δ receptors but not on the ± receptor, while β_h-(14–31) reduces the action of dynorphin on μ, δ and k receptors.     

Bicuculline actions on isolated rat atria, mouse vas-deferens and guinea-pig ileum are unrelated to GABA A receptor blockade

1. Some new pharmacological activities of bicuculline were found in isolated rat atria, mouse vas deferens and guinea-pig ileum. 2. In isolated rat atria bicuculline (10-300 microM) induced potent positive inotropic and negative chronotropic effects which were not antagonized by propranolol (1 microM), 6-hydroxydopamine pretreatment (50 mg/kg i.v. twice), ranitidine (3 microM) or atropine (1 microM). Bicuculline (10-300 microM) potentiated electrically evoked contractions in mouse vas deferens and inhibited them (30-500 microM) in guinea-pig ileum. It was inactive on unstimulated mouse vas deferens. 3. The above effects were completely reproduced by the bicuculline related-substance, beta-hydrastine, but not by the bicuculline N-methyl derivative, bicuculline methiodide (BMI), on the isolated rat atria. BMI inhibited instead of potentiating the mouse vas deferens twitches and potentiated instead of inhibiting the guinea-pig ileum twitches. 4. Picrotoxin, the other classic non-competitive GABA A antagonist, was completely devoid of the effects reported for bicuculline. 5. We concluded that, on the three preparations studied, bicuculline possesses some effects which are unrelated to its GABA A receptor blocking activity.     

Pharmacological characterisation of cannabinoid CB(1) receptors in the rat and mouse

The role of cannabinoid CB(1) receptors in sympathetic neurotransmission was characterised in nerve-mediated responses of isolated right atria, vasa deferentia and small mesenteric resistance arteries using the cannabinoid CB(1) receptor agonists Delta(9)-tetrahydrocannabinol, CP 55,940 and anandamide and the cannabinoid CB(1)-selective antagonist SR 141716A. In the mouse vas deferens, the twitch response was completely inhibited by each of the putative cannabinoid receptor agonists with pIC(50) values of CP 55,940, 9.2+/-0.1; Delta(9)-tetrahydrocannabinol, 8.4+/-0.1; anandamide, 7.1+/-0.1. SR 141716A 10-100 nM was a competitive antagonist of all three agonists with a pK(B) value of 8.4-8.6, consistent with an interaction at the cannabinoid CB(1) receptor. In the rat vas deferens CP 55,940 (0.01-10 microM) inhibited the contractions to a significant extent (88.5+/-0.5% at 10 microM; pIC(50) of 7.1+/-0.1) while Delta(9)-tetrahydrocannabinol and anandamide (both up to 10 microM) were inactive. CP 55,940 exhibited low potency in rat compared with mouse vas deferens and the rat concentration-response curve was not competitively antagonised by SR 141716A (100 nM) or SR 144528 (10 nM-10 microM), suggesting an interaction at a receptor(s) distinct from cannabinoid CB(1) or CB(2). Sympathetic nerve-induced tachycardia in rat and mouse atria, and rat mesenteric artery smooth muscle contractile responses to perivascular nerve stimulation, were not inhibited by Delta(9)-tetrahydrocannabinol, CP 55,940 or anandamide up to 1 microM. These data indicate that cannabinoid CB(1) receptor activation inhibits sympathetic neurotransmission only in the mouse vas deferens and thus point to species and regional differences in cannabinoid CB(1) receptor involvement in pre-synaptic inhibition of sympathetic neurotransmission and CP 55,940 may have inhibitory actions in rat vas deferens unrelated to cannabinoid receptor activity.     

CHANGES IN PREJUNCTIONAL POTENCY OF B-HT 933 DURING AGING IN THE RAT VAS DEFERENS

1. Age-related changes in prejunctional alpha 2-adrenoceptors were examined in the rat vas deferens using pharmacological techniques. 2. B-HT 933 (1 x 10(-8) - 1 x 10(-6) mol/L) caused a concentration-dependent inhibition of isometric contractions (tetrodotoxin-sensitive) induced by stimulation with single field-stimulus pulses, in both the epididymal and prostatic regions of rat vas deferens. The concentration-response curve to B-HT 933 was shifted to the right with age in the prostatic regions of the vas deferens. 3. In high concentrations (10(-6) - 3 x 10(-4) mol/L), B-HT 933 caused concentration-dependent enhancement of the contractile response to stimulation and evoked spontaneous contractile activity. No significant difference in this postjunctional activity occurred with age in either the prostatic or epididymal regions of the vas deferens. 4. Schild analysis revealed no significant differences in pA2 values for the antagonisms of the prejunctional inhibitory effect of B-HT 933 by rauwolscine in either the prostatic or epididymal regions of vas deferens between young and old rats. 5. These results could be interpreted as a decrease in alpha 2-adrenoceptor number with age. The more marked decrease in the prejunctional inhibitor potency of B-HT 933 in prostatic regions of vas deferens with aging may be due to a smaller receptor reserve in this region of the vas deferens.     

Synthesis and characterization of a selective peptide antagonist of neuropeptide Y vascular postsynaptic receptors.

1. A cyclic dimeric nonapeptide neuropeptide Y (NPY) receptor antagonist, 1229U91, was synthesized by Fmoc chemistry and dimerised in solution. Its effects were assayed in mesenteric arteries from rats and mice, and in rat vas deferens. 2. Mesenteric arteries were cannulated and pressurised to 55 mmHg and the external diameters continuously measured. NPY, PYY, Leu31Pro34NPY and NPY(13-36) each caused concentration-related contractions with the order of potency PYY > or = Leu31Pro34NPY = NPY > NPY (13-36), consistent with the Y1 receptor subtype. 3. 1229U91 had no agonist activity in the arteries but caused a concentration-related rightward shift of NPY (mouse arteries) or Leu31Pro34NPY (rat) concentration-response curves. The antagonism was competitive with pKBS of 7.69 +/- 0.15 and 7.47 +/- 0.13 in the mouse and rat arteries, respectively. 4. Sympathetic nerves in the vas deferens were stimulated with a single electrical field pulse every 20 s and the twitch responses recorded. NPY, PYY, Leu31Pro34NPY and NPY(13-36) inhibited the twitches with the order of potency PYY > NPY > NPY(13-36) >> Leu31Pro34NPY, consistent with the Y2 receptor subtype. 5. 1229U91 inhibited the vas deferens twitch with a shallow concentration-response curve and a time-course of inhibition distinct from that of NPY. 1229U91 (30 microM) did not cause a rightward shift of the NPY concentration-response curve. 1229U91 is at least 5 orders of magnitude less potent in the vas deferens than in rat brain Y2 binding assays reported by others, suggesting that the brain and vas deferens Y2 receptors are different. 6. It is concluded that 1229U91 is a competitive antagonist of NPY Y1 vascular receptors and has additional properties that inhibit the electrically evoked twitch of the rat vas deferens.     

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy.

The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the mu-opioid receptor selective agonist synthetic peptide (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at mu-opioid receptors in both organs. The -ol derivatives were slightly (2.3-4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the mu-opioid receptor pool in mouse vas deferens by 5x10(-7) M beta-funaltrexamine. The calculated receptor constants indicated a "high-affinity, low intrinsic efficacy" profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.     

P-7521--a new irreversible opioid ligand

In the receptor binding assay, P-7521 was a potent opioid ligand which acted mainly on mu receptor. The relative affinity ratio at mu, delta and kappa sites was 66:8:1. The inhibitory effects of P-7521 were 1868 and 6060 times more potent than morphine on the electrically evoked contractions in guinea pig ileum and mouse vas deferens, respectively and were readily antagonized by naloxone and Mr2266. These results indicate that P-7521 acted on mu receptor in guinea pig ileum and mouse vas deferens. In rabbit vas deferens, the compound had no agonist activity, but could antagonize the inhibitory effect of U-50488 H, a kappa agonist, showing the antagonistic characterization was on kappa receptor. The dissociation of P-7521 binding to opioid receptor were very difficult in mu binding assay and bioassays.     

In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pKi of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 microM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system.