Guanylate cyclase—Cyclic GMP in mouse cerebral cortex and cerebellum: Modification by anticonvulsants

Incubated tissue slices from mouse cerebral cortex and cerebellum readily accumulate cyclic GMP in response to a challenge by ouabain, NaN₃, NH₂OH, or KCl. Under similar conditions, l-glutamate, l-aspartate, glycine, γ-aminobutyric acid, kainic acid, and the calcium ionophore, A-23187, were ineffective. Inhibition of the ouabain-induced accumulation of cyclic GMP was evident with valproate, carbamazepine, clonazepam, phenytoin, and phenobarbital. Only phenytoin blocked the action of KCl and cyclic GMP responses to NaN₃ were inhibited by high concentrations of valproate, carbamazepine, phenytoin, or phenobarbital. The effects of NH₂OH were attenuated by high concentrations of carbamazepine, phenobarbital, and clonazepam (cortex only). Guanylate cyclase activity in homogenates of cortex and cerebellum was enhanced in the presence of NaN₃, NH₂OH, or Ca²⁺ (in low concentrations of Mn²⁺). The enzyme activation induced by Ca⁺ was blocked only by large (1 mm) amounts of carbamazepine. In like manner, large concentrations of carbamazepine, phenytoin (cortex), or clonazepam (cortex) were effective in reducing guanylate cyclase stimulation by NaN₃. No agent affected the NH₂OH responses. The results suggest that anticonvulsant drug actions with regard to central cyclic GMP systems are related to the Na⁺-induced depolarization of nerve tissue and not to any direct actions on the guanylate cyclase enzyme.     

Imaging LTP of presynaptic release of FM1-43 from the rapidly recycling vesicle pool of Schaffer collateral-CA1 synapses in rat hippocampal slices

Recent studies using the styryl dye FM1-43 and two-photon microscopy to directly visualize transmitter release at CA3-CA1 excitatory synapses in the hippocampus have demonstrated that activity-dependent long-term potentiation (LTP) and long-term depression are associated with alterations in vesicular release. It is not known whether particular vesicle pools preferentially express these alterations or what second messenger cascades are involved. To address these questions, we selectively loaded FM1-43 into the rapidly recycling pool (RRP) of vesicles by use of a brief hypertonic shock to release and load the RRP. We demonstrate here that the induction of LTP can lead to a selective long-lasting enhancement in presynaptic release from the RRP, while reserve pool kinetics remain unchanged. LTP of RRP release was N-methyl-d-aspartate receptor-dependent and also required production of the intercellular messenger NO and activation of receptor tyrosine kinase. Measurement of FM1-43 stimulus-evoked uptake rates following induction of LTP confirmed that LTP produces more rapid recycling of vesicles released by electrical stimulation, consistent with an enhanced release probability from the RRP.     

Plasma guanosine 3',5'-cyclic monophosphate and severity of peri/intraventricular haemorrhage in the preterm newborn

A poorly controlled cerebral circulation, caused by excessive production of nitric oxide, has been suggested as predisposing to peri/intraventricular haemorrhage (PIVH) in the immature neonate. It is hypothesized that a relation exists between plasma cyclic GMP (cGMP) as an effector of endogenous vasodilatory nitric oxide production and severity of PIVH. In 83 consecutively admitted preterm neonates, nitric oxide production was assessed by measuring plasma cGMP at 0, 24, 48, 72 and 168 h of age. Simultaneously, cranial ultrasound investigations were performed and haemodynamic parameters registered. The investigations showed that 60 neonates (72%) had no PIVH; 18 neonates (22%) had mild to moderate PIVH; and 5 neonates (6%) had severe PIVH. At 48 and 72 h of age, cGMP levels of infants with severe PIVH were significantly higher than those of infants with no or only mild PIVH, whereas at 72 and at 168 h, infants with moderate PIVHs had significantly higher cyclic cGMP levels than infants without PIVH. Finally, at 168 h of age infants with mild PIVH also had higher cyclic cGMP values than those of infants without PIVH. Maximal cGMP values preceded the final extension of PIVH in moderate and severe PIVHs. Blood pressure support was necessary significantly more often in infants with moderate and severe PIVH. A logistic regression model revealed that cGMP was significantly associated with PIVH, irrespective of gestational age, mean arterial pressure or severity of infant respiratory distress syndrome. Conclusion: Increased cGMP levels are associated with the development of PIVH. It is suggested that vasodilatory nitric oxide-induced impairment of cerebral autoregulation plays a role here.     

Pairing elevation of [cyclic GMP] with inhibition of PKA produces long-term depression of glutamate release from isolated rat hippocampal presynaptic terminals

Data suggest both presynaptic and postsynaptic changes contribute to activity-dependent long-term synaptic plasticity. We have shown that pairing elevation of intracellular [cyclic GMP], using the type V phosphodiesterase inhibitor zaprinast, with inhibition of cyclic AMP-dependent protein kinase (PKA), is sufficient to elicit chemical long-term depression (CLTD) of synaptic transmission at Schaffer collateral-CA1 and mossy fibre-CA3 synapses in rat hippocampus. CLTD does not require synaptic activity, and selective postsynaptic drug injections do not affect it, suggesting it is presynaptically induced and expressed. To directly evaluate this hypothesis, we tested whether CLTD of transmitter release can be expressed in isolated presynaptic nerve terminals. Presynaptic nerve terminals (synaptosomes) were isolated from rat hippocampi by Percoll density gradient centrifugation. Synaptosomes were loaded with [3H]glutamate, and basal and depolarisation-induced release of [3H]glutamate measured in control medium versus medium containing zaprinast (20 microm) plus or minus the PKA inhibitor H-89 (10 microm). Zaprinast produced a significant decrease in basal [3H]glutamate release. However, only combining zaprinast with H-89 significantly depressed K+-evoked [3H]glutamate release. After a 20-min drug washout, basal release returned to normal in all conditions, but K+-evoked [3H]glutamate release was persistently reduced only by the combination of zaprinast plus H-89. Long-term reduction of [3H]glutamate release from synaptosomes was completely prevented by the PKG inhibitor KT5823 (5 microm). These data demonstrate the existence of a presynaptic, cyclic GMP-PKG dependent cascade capable of expressing LTD of glutamate release from isolated hippocampal nerve terminals.     

人表皮生长因子的基础和应用研究

本文报道了有关人表皮生长因子（hEGF）及其受体（EGFR）的基础研究结果,以及在此基础上进行新药开发的有关工作;hEGF的基因合成、表达载体的构建和转化宿主细胞、表达产物的纯化和中试规模三批样品笔检验等的有关研究结果、hEGF滴眼液的动物实验和临床实验等新药研制过程。有关基础研究表明,编码51个氨基酸的hEGF可以在α因子前导肽的引导下在酵母体中分泌性表达,表达产物可促进角膜缘上皮细胞的增殖,可促进角膜碱烧伤的愈合,可用于口腔溃疡和皮肢烧伤的治疗,可对大鼠十二指肠溃疡有预防作用等,并用之制备了抗血清以测定血尿中EGF的浓度。研究表明EGF在大于10ng/ml浓度下对神经胶质瘤细胞株BT325和人阴道上皮癌细胞株A431的生长具有抑制作用,并用差异显示方法对此现象进行了分子机制的探讨。研究肾癌EGFR表达和DNA含量检测具有临床意义。对EGF的晶体进行了初步的结构分析。中试产品检测表明hEGF相对分子质量为6000、等电点为4．6、氨基端15个氨基酸顺序正确、具有EGF的免疫原性和生物活性。产品无酵母细胞的残余DNA。动物实验表明EFG在体内无蓄积,有明确的促进角膜上皮细胞增殖的作用,无急毒的长毒副作用,对眼无刺激和局部毒性作用。在四家医院进行的多中心双盲的临床实验以证实该滴眼液安全性、耐受量和有效性。在200例角膜移植和247例翼状胬肉切除术中有明显促进角膜损伤恢复的作用,其效果强于国外产阳性对照素高捷疗（Solcosery Eye Gel)。所有这些研制工作促成了利用酵母体系表达的人表皮生长因子制成的滴眼液获得了国家I类新药证书,这是我国首次批准的酵母体系表达的基因工程药物。     

Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells

Summary The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca²⁺]_i) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 M) enhanced the increase in [Ca²⁺]_i elicited by bradykinin (3 nM) and also caused an increase in resting [Ca²⁺]_i when given alone. This increase in resting [Ca²⁺]_i was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N^G-nitro-l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca²⁺]_i and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B₂-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B₂-receptors with a subsequent increase in [Ca²⁺]_i and nitric oxide formation. Send offprint requests to R. Busse at the above address     

宫清颗粒在药物流产中作用机制探讨

目的：探讨宫清颗粒在药物流产(药流)中的作用机制。方法：150例药流病人分为3组,每组50例。对照组单纯口服米非司酮与米索前列醇行药流,宫清颗粒Ⅰ组药流d1即加服宫清颗粒,宫清颗粒Ⅱ组于服米索前列醇2h后始加服宫清颗粒,观察用药前后血清绒毛膜促性腺激素β(β-HCG)、一氧化氮(NO)及血浆环鸟嘌呤核苷酸(cGMP)值变化。结果：药流后15d宫清颗粒Ⅰ,Ⅱ组β-HCG低于对照组[(2．0±s1．5),(2．9±1．9)μg·L~(-1)vs(5．1±2．4)μg·L~(-1)],差异均有显著意义(P<0．05),宫清颗粒Ⅰ组β-HCG低于宫清颗粒Ⅱ组(P<0．05)。药流后宫清颗粒Ⅰ组NO和cGMP值明显低于其他2组(P< 0．01)；药流后15d宫清颗粒Ⅰ,Ⅱ组NO和cGMP值均低于对照组(P<0．01)。结论：宫清颗粒通过下调血清β-HCG、影响血清NO、血浆cGMP含量而起作用,并具有协同米非司酮抗早孕的作用。     

Preferential potentiation by nitric oxide of spontaneous inhibitory postsynaptic currents in rat supraoptic neurones

Magnocellular neurones in the supraoptic nucleus and paraventricular nucleus express mRNA for nitric oxide synthase (NOS) and the expression becomes more prominent when the release of vasopressin or oxytocin is stimulated. It has also been reported that NO donors inhibit the electrical activity of supraoptic nucleus neurones, but the mechanism involved in the inhibition remains unclear. In the present study, to know whether modulation of synaptic inputs into supraoptic neurones is involved in the inhibitory effect of NO, we measured spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) from rat supraoptic nucleus neurones in slice preparations identified under a microscope using the whole-cell mode of the slice-patch-clamp technique. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reversibly increased the frequency of spontaneous IPSCs mediated by GABAA receptors, without affecting the amplitude, indicating that NO potentiated IPSCs via a presynaptic mechanism. The NO scavenger, haemoglobin, suppressed the potentiation of IPSCs by SNAP. On the other hand, SNAP did not cause significant effects on EPSCs mediated by non-NMDA glutamate receptors. The membrane permeable analogue of cGMP, 8-bromo cGMP, caused a significant reduction in the frequency and amplitude of both IPSCs and EPSCs. The results suggest that NO preferentially potentiates the inhibitory synaptic inputs into supraoptic nucleus neurones by acting on GABA terminals in the supraoptic nucleus, possibly via a cGMP-independent mechanism. The potentiation may, at least in part, account for the inhibitory action of NO on the neural activity of supraoptic neurones.