甾体化合物——LⅣ.化合物 S 醋酸酯经 Streptomyces roseochromogenus 的转化

Microbiological transformation of compound S acetate (I, R=Ac)to the corresponding 20β-hydroxy derivative(II,R=H),accompanied with hydrolysis of C₂1-acetate, was carried out by incubation with Streptomyces roseochromaogenus.     

β肾上腺素受体不可逆阻断剂BAlpM的合成

A highly potent beta-adrenergic irreversible antagonist——Bromoacetylalprenololmenthane (BAlpM)was synthesized by a Six step method with phenol and allychloride as the starting materials. Some improvement on purification of the product was described. The final product is identified by melting point, elemental analysis, UV and IR spectral analysis and mass spetrometry as well as β-adrenergic receptor bindingassays. [¹²⁵I]±IODOPINDOLOL binding assay of mouse lung cell membrane preparations treated with BAlpM in vitro or in vivo showed that there was a dosedependant decrease in the density of specific binding sites with no change in the Kd values. This result confirms that BAlpM is a β-adrenergic irreversible antagonist.     

甲基黄酮醇胺盐酸盐对β受体的阻断作用

The β-receptor blocking action of methylflavonolamine hydrochloride(MFA) was studied and compared with those of propranolol. The doseresponse curves of isoproterenol were shifted to the right by MFA on isolated rabbit atrium in this experiment. The pA₂ value and the slope of the regression line of MFA calculated from Schild plot were 5.53 and -0.84 respectively. The effects of MFA and propranolol on duck erythrocyte membranes were studied by the radioligand binding method. Both MFA and propranolol inhibited the binding of [³H] dihydroalprenolol to β-receptors. Their apparent equilibrium dissociation constants were 1.12×10^-5 mol/L and 5.50×10^-9 mol/L respectively. The affinity of propranolol to β-receptors of duck erythrocyte membranes was 2039-fold higher than that of MFA. These results demonstrates that MFA is a weak competitive β-receptor blocking agent.     

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 μM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 μM) inhibited desipramine-sensitive uptake of [³H] _{noradrenaline} (NA) (IC₅₀=97 μM). Saturation analysis showed that ketamine reduced V _max of [³H]NA uptake without changing K _m, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [³H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 μM) also inhibited the specific binding of [³H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [³H]desipramine binding revealed that ketamine increased K _d without altering B _max, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [³H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT. Received: 18 December 1997 / Accepted: 17 June 1998     

Δ16-娠烯-3β-乙酰氧基-11:20-二酮的极谱测定

A simple and rapid method for the determination of △~(16)—pregnene-3β—acetoxy — 11:20 — dione has been worked out. In an aqueous 40% iso-propanol solution of 0.1N NH₄Cl, this steroid gave a double polarographic wave which could be used for its determination. The current was measured at —1.50 v. vs. S.C.E. and was proportional to the concentration in the range of 36 to 180/μg/ml. From the experimental results, the diffusion current constant I and the diffusion coefficient D have been calculated to be 1.30 and 1.15 ×10^-6 cm²/sec. respectively. This method has been applied to the analysis of a sample; the result agreed with that obtained by the spectrophotometric method.     

5α-△9(11)-16β-甲基-3β,17α,21-三羟基-孕甾烯-3β,21-双醋酸酯-20酮和5α,17α-甲基-17β-羟基-雄甾-3酮的微生物转化

用诺卡氏菌与节杆菌混合菌种转化从蕃麻皂素制得的中间体5α-△⁹⁽¹¹⁾-16-16β-甲基-3β,17α,21三羟基孕甾烯-3β,21-双醋酸酯-20酮(Ⅰ)得50%的16β-甲基-△^1,4,9(11)-孕甾三烯-20酮(Ⅱ)和少量的16β-甲基-9,11α环氧-△¹,4孕甾二烯-20酮(Ⅲ)。另外,又用同样的混合菌种转化从剑麻皂素制得的中间体5α,17α甲基-17β羟基-雄甾-3酮(Ⅳ)得50%17α甲基-17β羟基-△^1,4-雄甾二烯-3酮(Ⅴ)。如改变培养基则得3,17β-羟基-17α-甲基-9酮基-9,10开环-1,3,5(10)雄甾三烯化合物。     

Na+-channel modulating effect of the inotropic compound S(-)BDF 9196 in human myocardium

S(-)BDF 9196, the active enantiomer of racemic (±)BDF 9148, has been shown to increase force of contraction in myocardium from different species including humans. The present study aimed to investigate the mechanism of the positive inotropic action of the active enantiomer S(-)BDF 9196 in human myocardium. In electrically driven human left ventricular papillary muscle strips (dilated cardiomyopathy, NYHA IV, cardiac transplantation, n=9), S(-)BDF 9196 increased force of contraction concentration-dependently. The maximal positive inotropic effect remained unchanged after the addition of carbachol (1 mmol/l), indicating a cAMP-independent mode of action of S(-)BDF 9196. While [³H]ouabain binding in human myocardial membranes was not influenced by S(-)BDF 9196 up to 10 μmol/l, the inward Na⁺-current in isolated human left ventricular myocytes was increased significantly by S(-)BDF 9196 (1 μmol/l, n=5). These results provide evidence that S(-)BDF 9196 increases force of contraction in human myocardium primarily by enhancing Na⁺-influx, while cAMP-dependent or Na⁺,K⁺-ATPase blocking effects do not seem to play a role. Received: 12 May 1998 / Accepted: 5 October 1998     

Na+ channel modulation and force-frequency relationship in human myocardium

The enhancement of force of contraction (FOC) following increasing frequencies of stimulation is an important mechanism of positive inotropy in human myocardium. The present study aimed to investigate the influence of alterations in Na⁺ influx on FFR in human myocardium. Isometric FOC of electrically stimulated right auricular trabeculae (AUT, n=12) from human nonfailing hearts (n=8) was measured at different stimulation rates (0.5-3 Hz) under control conditions, after increasing Na⁺ influx by the addition of (±)BDF 9148 (BDF, 3 μmol l^-1) and after decreasing Na⁺ influx by the addition of lidocaine (LIDO, 10 μmol l^-1). Additionally, the rate dependent changes in diastolic tension (DT) were measured in all experiments. Under control conditions FOC increased with increasing frequencies of stimulation. The rate at which maximal FOC was observed (SFmax) was 2.0±0.2 Hz and maximal increase in FOC (PIEmax) by increasing frequency of stimulation was +1.5±0.5 mN. After increase of Na⁺ influx by BDF (3 μmol l^-1) SFmax was decreased to 0.8±0.1 Hz (p<0.05 versus control) and PIEmax was +0.1±0.3 mN (p<0.05). When Na⁺ influx was diminished by LIDO (10 μmol l^-1) SFmax and PIEmax were increased compared to control (2.4±0.1 Hz and +4.1±0.9 mN, p<0.05 versus control). The diastolic tension (DT) of AUT at 3 Hz was not changed at higher rates in the control group and after application of LIDO (10 μmol l^-1), whereas after enhancement of Na⁺ influx by BDF there was an increase in DT of +0.7±0.2 at 3Hz (p<0.05 versus control and LIDO). An enhanced Na⁺ influx leads to a decrease in the optimal frequency and to a smaller force potentiation by higher stimulation rates which could be at least partly due to incomplete relaxation at higher frequencies, whereas a reduced Na⁺ influx is followed by opposite alterations. It is concluded that besides Ca²⁺ handling also Na⁺ influx and Na⁺ homeostasis might determine the frequency-induced force potentiation in human myocardium. Thus, the negative FFR in diseased human myocardium might result from changes in cellular Ca²⁺ or Na⁺ regulatory sites. Received: 20 November 1996 / Accepted: 21 February 1997     

16α-甲基-3β,17α-二羟基-5α-孕甾-20酮-3β-醋酸酯的微生物转化

16 α-Methyl-3 β,17 α-dihydroxy-5 α-pregnane-20-one-3 β-acetate (Ⅰ) was prepared from tigogenin and then incubated with a mixture of Mycobacterium smegmatis (MS₁) and Arthrobacter simplez (A₁) to give 16 α-methyl-17 α-hydroxy-△^{1, 4}-pregnadiene-3,20-dione (Ⅱ) with about 63% yield.Compound (Ⅱ) when incubated with Absidia glauca (AG₁) gave about 55% yield of 16 α-methyl-11 α, 17 α-dihydroxy-pregna-1,4-diene-3,20-dione (Ⅳ).     

Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery

The interaction of superoxide anions (O₂ ^–) generated by menadione with the synthesis and/or action of nitric oxide (NO), prostacyclin (PGI₂) and endothelium-derived hyperpolarizing factor (EDHF) was investigated in segments of the left anterior descending coronary artery (LAD) isolated from bovine hearts. EDHF and NO release were monitored by superfusion bioassay in segments pre-constricted with the thromboxane mimetic, U46619, PGI₂ release in addition by enzyme immunoassay for 6-keto-prostaglandin F_1α, and generation of O₂ ^– by lucigenin-enhanced chemiluminescence. Bradykinin (1–1,000 pmol) elicited a prominent, endothelium-dependent, relaxant response, 50–60% of which was insensitive to combined blockade of cyclooxygenase with diclofenac (1 μM) and NO synthase with N ^G-nitro-l-arginine (50 μM). This diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response was completely abrogated in the presence of tetrabutylammonium (3 mM), a non-selective inhibitor of Ca²⁺-dependent K⁺ channels, or when the segments were pre-constricted with potassium chloride (60 mM) instead of U46619, and thus most likely mediated by EDHF. Despite causing a two- to fourfold increase in the concentration of O₂ ^– in or at the vessel wall, menadione (30 μM) did not affect the diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response to bradykinin. When administered in the absence of N ^G-nitro-l-arginine, however, menadione significantly inhibited the relaxant response to bradykinin, presumably by attenuating the NO-mediated relaxation. Menadione also abolished the bradykinin-stimulated release of PGI₂ from luminally perfused segments of the LAD. This effect was more pronounced in the absence of N ^G-nitro-l-arginine, indicating that PGI₂ release in this preparation may be more sensitive to inhibition by peroxynitrite, the reaction product of NO and O₂ ^–, than to O₂ ^– alone. These findings suggest that, in contrast to NO and PGI₂, the release, and presumably also the mechanism of action, of EDHF in the bovine LAD is resistant to an increase in the local concentration of O₂ ^– or peroxynitrite which is thought to play an important role in coronary heart disease. Received: 27 August 1998 / Accepted: 18 November 1998     

Ontogeny of adenosine receptors in the longitudinal muscle and muscularis mucosae of the rat distal colon

The development of adenosine A₁ and A_2B receptors on the longitudinal muscle and muscularis mucosae of the neonatal rat distal colon has been investigated using homogenate binding, quantitative autoradiography and functional studies. In homogenate binding studies 1,3-[³H]-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) bound with high affinity to A₁ receptors in the muscularis mucosae and intact colon from rats aged 10, 15, 20, 25 and 30 days. The affinity of [³H]DPCPX was similar to that in the adult at all ages, but the density of binding sites was higher in the neonatal tissues. Quantitative autoradiography showed a higher density of [³H]DPCPX binding sites in the longitudinal muscle than in the muscularis mucosae at all ages studied (day 10 to adult), and this binding was concentration-dependently displaced by N ⁶-cyclopentyladenosine (CPA). In functional studies the longitudinal muscle relaxed in response to 5’-N-ethylcarboxamidoadenosine (NECA) and CPA at all ages studied (15–30 days), NECA being more potent than CPA. The potency of NECA remained constant and it was antagonised by 1 μM DPCPX at all ages with pA ₂-values consistent with activation of A₂ receptors. The inactivity of 2-[p-(carboxyethyl)-phenylethylamino]-5’-N-ethylcarbox-amidoadenosine (CGS 21680) indicated that the A₂ receptors were of the A_2B subtype. The muscularis mucosae contracted in response to CPA at all ages studied (day 15 to adult) and the antagonism by DPCPX (10 nM) were consistent with activation of A₁ receptors. In conclusion, binding, autoradiographic and functional studies identified A₁ receptors on the rat colon muscularis mucosae at all ages studied. However, while binding and autoradiographic localisation showed the presence of A₁ receptors in the longitudinal muscle at all ages studied, functional data only revealed the presence of A_2B receptors. Received: 3 July 1998 / Accepted: 25 November 1998     

Local administration of Δ9-tetrahydrocannabinol attenuates capsaicin-induced thermal nociception in rhesus monkeys: a peripheral cannabinoid action

  Rationale: Cannabinoids can reduce nociceptive responses by acting on peripheral cannabinoid receptors in rodents. Objectives: The study was conducted to evaluate the hypothesis that local administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) can attenuate capsaicin-induced nociception in rhesus monkeys. Methods: Capsaicin (100 μg) was applied locally in the tail of rhesus monkeys to evoke a nociceptive response, thermal allodynia, in normally innocuous 46°C water. Δ⁹-THC (10–320 μg) was coadministered with capsaicin in the tail to assess local antinociceptive effects. In addition, a local antagonism study was performed to confirm the selectivity of Δ⁹-THC action. Results:Δ⁹-THC dose-dependently inhibited capsaicin-induced allodynia. This local antinociception was antagonized by small doses (10–100 μg) of the cannabinoid CB₁ antagonist, SR141716A, applied in the tail. However, 100 μg SR141716A injected subcutaneously in the back did not antagonize local Δ⁹-THC. Conclusions: These results indicate that the site of action of locally applied Δ⁹-THC is in the tail. It provides functional evidence that activation of peripheral cannabinoid CB₁ receptors can attenuate capsaicin-induced thermal nociception in non-human primates and suggests a new approach for cannabinoids in pain management. Received: 1 December 1998 / Final version: 13 January 1999     

In vitro studies on the mechanism by which (+)-norfenfluramine induces serotonin and dopamine release from the vesicular storage pool

(+)-Norfenfluramine is the main metabolite of the serotoninergic anorectic agent (+)-fenfluramine. Both compounds inhibit 5-HT reuptake and stimulate its release, although they induce release from different pools, with (+)-norfenfluramine acting primarily on the cytoplasmic pool. Moreover, (+)-norfenfluramine was more potent than the parent drug in inducing dopamine release. In order to investigate whether (+)-norfenfluramine induces a Ca²⁺-dependent vesicular release, like some amphetamine derivatives, in the present study we preloaded synaptosomes with the [³H]neurotransmitter ([³H]5-HT or [³H]dopamine), superfused (washed) them for 47 min in the absence of pargyline and then exposed them to the releasing stimulus. With this protocol, the cytoplasmic pool should be absent and the [³H]neurotransmitter should mainly be stored in synaptic vesicles, where (+)-norfenfluramine should act to induce release. This was confirmed by a significant decrease of (+)-norfenfluramine-induced [³H]5-HT and [³H]dopamine release after reserpine pretreatment. The dose-response curves of (+)-norfenfluramine-induced [³H]5-HT release were superimposable in hippocampus and hypothalamus, and also superimposable on the curve for (+)-fenfluramine-induced [³H]5-HT release; the dopamine releasing potency of (+)-norfenfluramine in the striatum was more than ten times lower. The [³H]5-HT release induced by (+)-norfenfluramine was partly (about 50%) but significantly Ca²⁺-dependent, and it was also markedly (68%) inhibited by Cd²⁺, a non-specific blocker of voltage-dependent Ca²⁺ channels, suggesting that the Ca²⁺-dependent release is mediated by entry of Ca²⁺ into the synaptosomes through these channels. The [³H]dopamine release induced by 5 μM (+)-norfenfluramine was completely Ca²⁺-independent whereas at higher concentrations (10 and 20 μM) it was only slightly (20%) Ca²⁺-dependent. We have no clear explanation why (+)-norfenfluramine has these different effects on serotoninergic and dopaminergic synaptosomes. Received: 6 April 1998 / Accepted: 9 June 1998     

红花黄素对高血压大鼠的降压作用及对肾素-血管紧张素的影响

Safflower yellow (SY) is a mixture of chalconoid compounds extracted from Carthamus tinctorius L. Ig SY 1～2 g·kg^-1·d^-1lowered the blood pressure of spontaneously hypertensiverats (SHR), for about 1.86～3.86 kPa. Five weeks after administration of SY, the plasma renin ac-tivity and angiotensin Ⅱ level diminished in the SHR experimental groups. These suggest that the de-crease of blood pressure is mediated by the renin-angiotensin system.     

Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current

The putative coupling between stably expressed recombinant h 5-HT_1B or h 5-HT_1D receptors and K⁺ channels which regulate excitability was investigated in C6 glioma cells. Outward K⁺ currents (I _K) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-HT_1B or h 5-HT_1D receptors using the patch-clamp technique in the whole-cell configuration. I _K was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-HT_1B or h 5-HT_1D receptors, sumatriptan similarly increased I _K in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with EC₅₀ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in I _K in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT_1B and h 5-HT_1D receptors in transfected C6 cells. In the presence of the mixed 5-HT_1B/D receptor antagonist GR 127935 (0.1 μM), sumatriptan (1 μM) failed to significantly increase I _K in C6 cells expressing h 5-HT_1B receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 μM) was required to significantly inhibit sumatriptan-evoked increases in I _K in C6 cells expressing h 5-HT_1D receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT_1B and h 5-HT_1D receptors, respectively. In C6 cells expressing either cloned h 5-HT_1B or h 5-HT_1D receptors, sumatriptan-induced increases in I _K were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _K in the presence of dibutyryl cAMP (10 μM) or when a nominally Ca²⁺-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the Ca²⁺-dependent K⁺ channel blockers iberiotoxin (0.1 μM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in I _K (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-HT_1B receptors, confirming the involvement of Ca²⁺-dependent K⁺ channels. In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _k after 30-min incubation with thapsigargin (1 μM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-HT_1B and h 5-HT_1D receptors stably transfected in C6 glioma cells are positively coupled to Ca²⁺-dependent K⁺ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP₃ receptor-mediated intracellular Ca²⁺ release. Received: 15 April 1998 / Accepted: 9 September 1998     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

鲜罗汉果中黄酮甙的分离及结构测定

A new flavonol glycoside named grosvenorine and a known compoundwere isolated from the fresh fruits of Siraitia grosvenori( swingle)C. By means of UV,FAB-MS,¹H-¹H COSY,¹³C-1H COSY and NOE difference ,spectra,the structure of grosvenorinewas established as kaempferol-3-O-α-L-rhafmnopyranoside-7-O[β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside].The known compound was identified as kaempferol-3,7-α-L-dirhamnopyra-noside.     

Nociceptin inhibits noradrenaline release in the mouse brain cortex via presynaptic ORL1 receptors

A fourth type of opioid receptor, termed ORL₁, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [³H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca²⁺ 1.3 mM into Ca²⁺-free K⁺-rich (15 mM) medium. Nociceptin 1 μM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 μM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 μM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC₅₀ 7.5) was shifted to the right by the non-selective ORL₁ receptor antagonist naloxone benzoylhydrazone and the selective ORL₁ receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ (pA₂ 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ caused an inhibition by maximally 35% (pEC₅₀ 7.0; intrinsic activity α 0.45). The inhibitory effect of [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca ²⁺ -evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL₁ receptors, which are located presynaptically on noradrenergic neurones. Received: 19 June 1998 / Accepted: 17 July 1998