Role of GABA B receptors in the endomorphin-1-, but not endomorphin-2-, induced dopamine efflux in the nucleus accumbens of freely moving rats

In vivo microdialysis was used to study the effects of the locally applied GABA B receptor antagonist 2-hydroxysaclofen and GABA B receptor agonist baclofen on the basal dopamine efflux as well as on the endomorphin-1- and endomorphin-2-induced dopamine efflux in the nucleus accumbens of freely moving rats. 2-Hydroxysaclofen (100 and 500 nmol) increased basal dopamine efflux. Baclofen (2.5 and 5 nmol) failed to affect basal dopamine efflux. 2-Hydroxysaclofen (1 and 10 nmol) which did not alter the basal dopamine efflux, enhanced the endomorphin-1 (25 nmol)-induced dopamine efflux. Baclofen (2.5 and 5 nmol) failed to affect endomorphin-1 (25 nmol)-induced dopamine efflux, but it counteracted the 2-hydroxysaclofen-induced increase of the endomorphin-1-elicited dopamine efflux. Neither 2-hydroxysaclofen (10 nmol) nor baclofen (5 nmol) affected the endomorphin-2 (25 nmol)-induced dopamine efflux. The doses mentioned are the total amount of drug over the infusion period that varied across the drugs (25 or 50 min). These results suggest that accumbal GABA B receptor plays an inhibitory role on the basal as well as the endomorphin-1-elicited accumbal dopamine efflux. The present results support our earlier reported notion that endomorphin-1 and endomorphin-2 increase accumbal dopamine efflux by different mechanisms. Finally, it is suggested that a decrease of endogenous accumbal GABA reduces the accumbal GABA B receptor-mediated GABA-ergic inhibition, enhancing thereby the accumbal dopamine efflux.     

强啡肽A（1－17）对大鼠脊髓组织钙调蛋白含量和磷酸二酯酶活性的影响

观察强啡肽Ａ（１－１７）经大鼠蛛网膜下腔注射后对胞内Ｃａ２＋受体钙调蛋白（ＣａＭ）含量及其依赖于Ｃａ２＋／ＣａＭ的磷酸二酯酶（ＰＤＥ）活性的影响。结果表明：强啡肽Ａ（１－１７）１０、２０ｎｍｏｌ给药１０ｍｉｎ可使脊髓组织ＣａＭ含量和ＰＤＥ活性明显下降，呈量效依赖关系，２ｈ后均有不同程度的恢复。选择性ｋ型阿片受体拮抗剂ｎｏｒ－ＢＮＩ３０ｎｍｏｌ、兴奋性氨基酸ＮＭＤＡ受体特异性拮抗剂ＡＰＶ１０ｎｍｏｌ可显著对抗强啡肽Ａ（１－１７）２０ｎｍｏｌ降低脊髓组织ＣａＭ含量的作用并完全阻断强啡肽Ａ（１－１７）对ＰＤＥ活性的抑制；Ｌ型Ｃａ２＋通道阻断剂异搏定１００ｎｍｏｌ亦可部分阻断强啡肽Ａ（１－１７）２０ｎｍｏｌ对脊髓组织ＣａＭ含量和ＰＤＥ活性的影响。     

Effects of morphine and endomorphins on the polysynaptic reflex in the isolated rat spinal cord

At the spinal level, -opioids exert their actions on nociceptive primary afferent neurons both pre- and postsynaptically. In the present study, we used an in vitro isolated neonatal rat (11–15 days old) spinal cord preparation to examine the effects of morphine and the endogenous -opioid ligands endomorphin-1 (EM-1) and endomorphin-2 (EM-2) on the polysynaptic reflex (PSR) of dorsal root-ventral root (DR-VR) reflex. The actions of -opioids on spinal nociception were investigated by quantification of the firing frequency and the mean amplitude of the PSR evoked by stimuli with 20×threshold intensity. EM-1 decreased the mean amplitude of PSR, whereas EM-2 and morphine decreased the firing frequency. The pattern of the effects elicited by morphine was the same as that for EM-2, except at high concentration. Naloxonazine, a selective ₁ opioid receptor antagonist, had no significant effect on PSR by itself, but blocked the inhibition of PSR firing frequency or amplitude induced by EM-1, -2 and morphine. This may suggest that EM-1, EM-2 and morphine modulate spinal nociception differently and act mainly at the ₁-opioid receptors. Although they all act via ₁-opioid receptors, their different effects on the PSR may suggest the existence of different subtypes of the ₁-opioid receptor. The present data is also consistent with a further hypothesis, namely, that morphine and EM-2 activate a subtype of ₁-opioid receptor presynaptically, while EM-1 acts mainly through another subtype postsynaptically. However, since other reports indicate that EM-2, but not EM-1, could stimulate the release of enkephalins or dynorphin, presynaptic and receptors may be also involved indirectly in the different regulation by -opioids at the spinal level.     

Centrally administered histamine evokes the adrenal secretion of noradrenaline and adrenaline by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats

Plasma adrenaline is originated from adrenal medulla, while plasma noradrenaline reflects the release from sympathetic nerves in addition to the secretion from adrenal medulla. The present study was designed to characterize the source of plasma catecholamines induced by centrally administered histamine, with regard to the brain prostanoids. Intracerebroventricularly (i.c.v.) administered histamine (1, 5 and 10 microg/animal) elevated plasma noradrenaline and adrenaline (noradrenaline相似文献   

Antagonistic cannabinoid CB1/dopamine D2 receptor interactions in striatal CB1/D2 heteromers. A combined neurochemical and behavioral analysis

In vitro results show the ability of the CB(1) receptor agonist CP 55,940 to reduce the affinity of D(2) receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D(2) receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB(1)-D(2) receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB(1)/D(2) receptor-receptor interactions may occur in CB(1)/D(2) formed heteromers. Antagonistic CB(1)/D(2) interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB(1) receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB(1) receptor antagonist rimonobant but also the specific A(2A) receptor antagonist MSX-3 blocked the inhibitory effect of CB(1) receptor agonist on D(2)-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB(1)/D(2) receptor-receptor interactions within CB(1)/D(2) heteromers in which A(2A) receptors may also participate.     

Glomeruli and microvessels of the rabbit kidney contain both A1- and A2-adenosine receptors

Summary Rabbit renal cortices were fractionated by collagenase dispersion and glomeruli, microvessels and tubuli purified on a discontinuous sucrose gradient. Binding experiments with (–)[¹²⁵I]N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) provided evidence for the presence of A₁-adenosine receptors in the glomerular and microvascular fraction. With glomeruli, saturation isotherms for specific [¹²⁵I]HPIA binding were mono-phasic with a K _D of 1.3 nmol/l and a B _maxof 7.7 fmol/mg protein. In kinetic experiments, an association rate constant of 4.9 × 105 (mol/ 1)^–1 s^–1 and a dissociation rate constant of 4.3 × 10^–4 s^–1 were obtained, yielding a K _D of 0.9 nmol/l. Adenosine analogs displaced [¹²⁵I]HPIA binding with a rank order of potency typical of A₁-adenosine receptors; furthermore, binding was inhibited by methylxanthines and modulated by GTP. Saturation experiments with the microvessels revealed a K _D of 1.9 nmol/l and a B _max of 13.4 fmol/mg protein. However, no inhibition of glomerular and microvascular adenylate cyclase activity could be demonstrated, but instead both 5-N-ethylcarboxamido-adenosine (NECA) and N⁶-(R-phenylisopropyl)-adenosine (R-PIA) stimulated enzyme activity, with EC₅₀ values of 0.14 mol/l and 1.5 mol/l, respectively. The concentration-response curve for NECA was shifted to the right (factor 9) by 10 mol/l 8-phenyltheophylline. On the other hand, computer simulation of biphasic curves (adenylate cyclase inhibition in the presence of activation via a stimulatory receptor) indicates that the failure to observe an A₁-adenosine receptor-mediated inhibition of adenylate cyclase activity in the presence of stimulatory adenosine receptors may be attributable to methodological constraints. The results demonstrate that both A₁- and A₂-adenosine receptors are present in rabbit glomeruli and microvessels. It is suggested that both receptors are involved in the control of renin secretion.Abbreviations R-PIA (–)N⁶(R-phenylisopropyl)-adenosine - NECA 5-(N-ethyl-carboxamido)-adenosine - S-PIA (+)N⁶-(S-phenylisopropyl)-adenosine - I-HPIA (–)N⁶-(3-iodo-4-hydroxy-phenylisopropyl)-adenosine - HPIA (–)N⁶-(4-hydroxyphenylisopropyl)-adenosine - [¹²⁵I]HPIA (–)N6-(3-[¹²⁵I]iodo-4-hydroxy-phenylisopropyl)-adenosine - ATP adenosine-5-triphosphate - cAMP cyclic 3,5-adenosine-monophosphate - GTP guanosine-5-triphosphate - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA (ethylenedinitrilo)-tetraacetic acid Send offprint requests to W. Schütz at the above addressThis study was supported by the Fonds zur Förderung der wissenachiftlichen Forschung in Österreich (Project 5712)     

Differential contributions of adenosine to hypoxia-evoked depressions of three neuronal pathways in isolated spinal cord of neonatal rats

BACKGROUND AND PURPOSE

Hypoxic effects on neuronal functions vary significantly with experimental conditions, but the mechanism for this is unclear. Adenosine has been reported to play a key role in depression of neuronal activities in the CNS during acute hypoxia. Hence, we examined the effect of acute hypoxia on different spinal reflex potentials and the contribution of adenosine to them.

EXPERIMENTAL APPROACH

Spinal reflex potentials, monosynaptic reflex potential (MSR), slow ventral root potential (sVRP) and dorsal root potential (DRP), were measured in the isolated spinal cord of the neonatal rat. Adenosine release was measured by using enzymatic biosensors.

KEY RESULTS

In the spinal cord preparation isolated from postnatal day 5–8 rats at 27°C, acute hypoxia induced adenosine release and depressed three reflex potentials. However, in postnatal day 0–3 rats at 27°C, the hypoxic-induced adenosine release and depression of MSR were negligible, while the depression of sVRP and DRP were perceptible responses. In postnatal day 0–3 rats at 33°C, hypoxia evoked adenosine release and depression of MSR. An adenosine A₁ receptor selective antagonist and a high [Ca²⁺]_o, which suppressed adenosine release, abolished the hypoxic-induced depression of MSR but not those of sVRP and DRP.

CONCLUSIONS AND IMPLICATIONS

Hypoxic-induced depression of MSR depends on adenosine release, which is highly susceptible to age, temperature and [Ca²⁺]_o. However, a large part of the depressions of DRP and sVRP are mediated via adenosine-independent mechanisms. This differential contribution of adenosine to depression is suggested to be an important factor for the variable effects of hypoxia on neuronal functions.     

Effects of kinin B1 and B2 receptor antagonists on overactive urinary bladder syndrome induced by spinal cord injury in rats

Background and Purpose

Kinin B₁ and B₂ receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive.

Experimental Approach

We investigated the role of kinin B₁ and B₂ receptors in OAB after SCI in rats.

Key Results

SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B₁ receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B₁ protein in the urinary bladder and B₁ and B₂ receptor protein in spinal cord. Interestingly, both B₁ and B₂ protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B₁ or B₂ agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B₂ selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B₁ antagonist des-Arg⁹-[Leu⁸]-bradykinin reduced the number of NVCs while the non-peptide B₁ antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume.

Conclusions and Implications

Taken together, these data show the important roles of B₁ and B₂ receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.     

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Role of spinal serotonin1 receptor subtypes in thermally and mechanically elicited nociceptive reflexes

The ability of 5-HT_1A and 5-HT_1B agonists to alter a spinal animal's nociceptive threshold was examined using two analgesiometric tests. In the spinal withdrawal reflex test, administration of the selective 5-HT_1A agonists ipsapirone, gepirone and PAPP resulted in significant dose-dependent increases in receptive field (RF) area for withdrawal reflexes when compared to predrug baseline values, indicating an increase in nociceptive sensitivity. The average overall percent maximal increase in RF area following administration of 5-HT_1A selective compounds was: 80±16% for the ventroflexion reflex, 90±6% for the dorsiflexion reflex and 87±8% for the lateral flexion reflex. Similar to the effects noted with 5-HT_1A agonists, administration of 5-HT_1B agonists RU24969, mCPP and TFMPP resulted in a hyperalgesic response with an overall percent maximal increase of 43±6% for the ventroflexion reflex, 51±6% for the dorsiflexion reflex and 38±9% for the lateral flexion reflex. In the tail-flick analgesiometric test, administration of the 5-HT_1A agonists 8-OH-DPAT and ipsapirone and the 5-HT_1B agonists RU24969 and mCPP resulted in a significant dose-dependent increase in tail-flick latencies when compared to predrug baseline values, indicating a decrease in nociceptive sensitivity to noxious thermal stimuli. No differences in magnitude of the effect of the two receptor subtypes were found, indicating that stimulation of either 5-HT_1A or 5-HT_1B receptors was equipotent in producing the antinociceptive tail-flick response. Administration of the 5-HT_1A antagonist meter-goline completely reversed the increase in TFL produced by RU24969, providing further evidence that the effects seen here are mediated by spinal 5-HT receptors. The results of this study indicate that spinal 5-HT₁ receptor subtypes may either facilitate or inhibit nociceptive input depending upon the type of nociceptor that is activated, as opposed to the type of receptor subtype that is stimulated.     

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist,directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT_1A receptor agonist/5-HT_2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1–10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1–1000 g/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1–10 g/kg and 0.1–3 g/kg i.v. respectively, and reduced it at higher doses, 30–300 g/kg and 10–30 g/kg i.v., respectively.The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT_1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 g/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OHDPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.These findings suggest that BIMT 17 directly inhibits the electrical activity of medial prefronto-cortical neurons through its dual mode of receptor interaction.     

Pharmacological studies of 8-OH-DPAT-induced pupillary dilation in anesthetized rats

Serotonin (5-HT)(1A) receptor agonists have been reported to produce mydriasis in mice, and miosis in rabbits and humans. However, the underlying mechanisms for this action are unclear. This study was undertaken in an attempt to explore the mechanism by which 5-HT(1A) receptors are involved in the modulation of pupillary size in pentobarbital-anesthetized rats. Intravenous administration of the 5-HT(1A) receptor agonist, (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.003-3 mg/kg), elicited dose-dependent pupillary dilation, which was not affected by section of the preganglionic cervical sympathetic nerve. 8-OH-DPAT-elicited mydriatic responses were attenuated by the selective 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100635; 0.3-1 mg/kg, i.v.), as well as by the selective alpha(2)-adrenoceptor antagonist, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine hydrochloride (RS 79948; 0.3 mg/kg, i.v.), but not by the selective alpha(1)-adrenoceptor antagonist, prazosin (0.3 mg/kg, i.v.). Mydriatic responses elicited by the alpha(2)-adrenoceptor agonist, guanabenz (0.003-0.3 mg/kg, i.v.), were not antagonized by WAY 100635 (0.3-1 mg/kg, i.v.). To determine whether central nervous system (CNS) 5-HT(1A) receptors, like alpha(2)-adrenoceptors, are involved in reflex mydriasis, voltage response curves of pupillary dilation were constructed by stimulation of the sciatic nerve in anesthetized rats. WAY 100635 (1 mg/kg, i.v.) did not antagonize the evoked reflex mydriasis, which, however, was blocked by RS 79948 (0.3 mg/kg, i.v.). Taken together, these results suggest that 8-OH-DPAT produces pupillary dilation in anesthetized rats by stimulating CNS 5-HT(1A) receptors, which in turn trigger the release of norepinephrine, presumably from the locus coeruleus. The latter reduces parasympathetic neuronal tone to the iris sphincter muscle by stimulation of postsynaptic alpha(2)-adrenoceptors within the Edinger-Westphal nucleus. Unlike alpha(2)-adrenoceptors, 5-HT(1A) receptors in the CNS do not mediate reflex mydriasis evoked by sciatic nerve stimulation.     

Fasudil促进大鼠损伤脊髓神经功能恢复的研究

目的研究Rho激酶抑制剂Fasudil对大鼠脊髓损伤的促修复作用。方法用健康成年SD大鼠建立脊髓损伤模型,通过腹腔注射盐酸法舒地尔,并设立对照。术后1、2、4周,运用BBB功能评分进行后肢运动功能评价;损伤后4周,脊髓损伤局部行SABC法ROCK2免疫组织化学染色。结果术后1、2、4周,Fasudil治疗组与对照组比较,评分明显增高(P<0.05);术后4周Fasudil治疗组损伤局部组织内ROCK2的表达较对照组明显减少(P<0.05)。结论腹腔注射Fasudil能够减少脊髓损伤局部ROCK2表达,改善大鼠的运动功能。     

N-乙酰半胱氨酸对大鼠急性脊髓损伤后继发性脊髓损伤的保护作用

目的 探讨N-乙酰半胱氨酸(NAC)对大鼠脊髓急性损伤后继发性脊髓损伤的保护机制.方法 成年SD大鼠18只随机均分成单纯椎板切除(对照组)、急性脊髓损伤(损伤组)和急性脊髓损伤后NAC治疗(治疗组)三组.用30 g力量动脉瘤夹从两侧夹闭脊髓30 s建立脊髓损伤模型.治疗组术后15 min、1、2和3d分别予NAC 150 mg/kg腹腔注射;对照组和损伤组腹腔注射等量生理盐水.所有动物于术后3d处死取材,用凝胶电泳迁移率分析和免疫组化检测脊髓组织中核因子κB(NF-κB),ELISA法检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6表达.结果 损伤组脊髓组织中NF-κB、TNF-α、IL-1β和IL-6水平均比对照组升高(P＜0.05),治疗组脊髓组织中各因子水平均比损伤组显著降低(P＜0.05).结论 NAC可以通过抑制大鼠急性脊髓损伤后NF-κB的活性,进一步下调TNF-α、IL-1β和IL-6的表达,从而减轻继发性炎症反应所导致的脊髓损伤.     

Discovery of Pyridone-Substituted Triazolopyrimidine Dual A2A/A1 AR Antagonists for the Treatment of Ischemic Stroke

Ischemic stroke is a complex systemic disease characterized by high morbidity, disability, and mortality. The activation of the presynaptic adenosine A_2A and A₁ receptors modifies a variety of brain insults from excitotoxicity to stroke. Therefore, the discovery of dual A_2A/A₁ adenosine receptor (AR)-targeting therapeutic compounds could be a strategy for the treatment of ischemic stroke. Inspired by two clinical phase III drugs, ASP-5854 (dual A_2A/A₁ AR antagonist) and preladenant (selective A_2A AR antagonist), and using the hybrid medicinal strategy, we characterized novel pyridone-substituted triazolopyrimidine scaffolds as dual A_2A/A₁ AR antagonists. Among them, compound 1a exerted excellent A_2A/A₁ AR binding affinity (K_i = 5.58/24.2 nM), an antagonistic effect (IC₅₀ = 5.72/25.9 nM), and good metabolic stability in human liver microsomes, rat liver microsomes, and dog liver microsomes. Importantly, compound 1a demonstrated a dose–effect relationship in the oxygen-glucose deprivation/reperfusion (OGD/R)-treated HT22 cell model. These findings support the development of dual A_2A/A₁ AR antagonists as a potential treatment for ischemic stroke.     

Squalestatin protects neurons and reduces the activation of cytoplasmic phospholipase A2 by Abeta(1-42)

Alzheimer's disease is characterised by the loss of neurons and the production of Abeta peptides. We report that the addition of Abeta(1-42) to neurons resulted in activation of cytoplasmic phospholipase A(2) (cPLA(2)), the production of prostaglandin E(2), synapse damage and reduced neuronal survival. Pre-treatment with simvastatin, a clinically relevant statin that penetrates the brain, protected against Abeta(1-42) induced synapse damage and neuronal death in vitro. The neuroprotective effects of simvastatin were shared by squalestatin, a squalene synthase inhibitor that reduces neuronal cholesterol production and crucially, does not affect isoprenoid formation. The protective effect of both these drugs was reversed by the addition of exogenous cholesterol. These drugs did not alter the amounts of extracellular Abeta(1-42) ingested by neurons; rather they reduced Abeta(1-42) induced activation of cPLA(2) and prostaglandin E(2) production. Treatment prevented the migration of Abeta(1-42) and cPLA(2) to caveolin-1 containing lipid rafts. We propose that critical concentrations of Abeta(1-42) trigger the amalgamation of individual micro-domains containing signalling molecules to form lipid raft platforms in which sustained activation of cPLA(2) leads to neuronal dysfunction and ultimately neuronal death. This process is dependent on the amounts of cholesterol in neuronal membranes and is susceptible to treatment with squalestatin or simvastatin.     

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.     

Endomorphin-1 and endomorphin-2 activate µ-opioid receptors in myenteric neurons of the guinea-pig small intestine

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central μ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 μM) inhibited (–log EC₅₀ values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective μ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), caused a concentration-dependent (30 nM–1 μM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA ₂ values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate μ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists. Received: 6 August 1998 / Accepted: 5 October 1998