再生障碍性贫血患者外周血单个核细胞产生某些细胞因子能力的变化及意义

观察再生障碍性贫血患者外周血单个核细胞（ＰＢＭＮＣ）产生 ＩＬ－１α、ＩＬ－２的能力及外周血清ｓＩＬ－２Ｒ水平,探讨这些变化的意义。方法：应用ＥＬＩＳＡ法测定细胞因子活性。结果〔显示再障患者ＰＢＭＮＣ产生ＩＬ－１α的能力明显低于对照组（Ｐ＜０．０１）;而产生 ＩＬ－２的能力则明显高于对照组（Ｐ＜０．０１）;血清 ｓＩＬ－２Ｒ水平明显高于对照组（Ｐ＜０．０１）。结论：这三项检测结果对于认识再障贫血的发病机制、辅助诊断疾病的严重程度、指导治疗和估计预后有一定意义。     

Hypozincaemia in febrile convulsion

To understand further the role of trace elements in the pathogenesis of febrile convulsions, serum zinc (Zn), copper (Cu), magnesium (Mg) and CSF Zn, Cu, Mg and protein levels were measured by spectrometry in patients with febrile convulsion (n=19), bacterial meningitis (n=9), viral CNS infection (n=16) and in the control groupn=10) which consisted of children with signs of meningeal irritation due to upper respiratory tract infection but normal CSF findings. Samples were obtained within 6 h after admission to hospital. Mean serum and CSF Zn levels in the febrile convulsion group were significantly lower than in the other groups (for serum Zn: 0.66±0.03 mg/l vs 0.98±0.07 mg/l, 1.06±0.08 mg/l, 1.05±0.09 mg/lP<0.05; for CSF Zn: 22.96±1.62 g/l vs 75.47 ±6.9 g/l, 50.32±5.235 g/l, 39.85 ±2.81 g/lP<0.05). A linear relationship was established between serum Zn and CSF Zn levels (P<0.001). Mean CSF Zn, Cu and protein levels in the bacterial meningitis group were significantly higher than in the other groups (for CSF Cu 63.94±6.33 g/l vs 38.77±2.70 g/l, 35.84±3.48 g/l, 33.86±2.88 g/lP<0.05; for CSF protein 0.80 ± 0.12 g/l vs 0.22±0.02 g/l, 0.53±0.08 g/l, 0.19±0.01 g/lP<0.05). In children with meningitis, the elevation of the mean CSF Zn and Cu levels may result from the breakdown of the blood-brain barrier and subsequent leakage of trace elements and protein from serum to CSF. There was no significant difference between the four groups in terms of mean serum Mg and mean CSF Mg levels.Conclusion Serum and CSF Zn levels are decreased in children with febrile seizures. Zinc deprivation may play a role in the pathogenesis of febrile seizures.     

Desensitization of the adipocyte A(1) adenosine receptor during untreated experimental diabetes mellitus

We examined the changes that occur in the adenosine receptor system during diabetes mellitus. Experimental diabetes mellitus was induced in male Lewis rats with streptozocin (65 mg/kg), and A₁ adenosine receptor binding was characterized with [¹²⁵I]N ⁶-2-(4-aminophenyl) ethyladenosine. In adipocytes, high-affinity A₁ adenosine receptor binding decreased from 1466±228 of protein to 312±123 fmol/mg of protein (p<0.01) following 14 d of untreated diabetes mellitus. Neither the dissociation constant (K _d=1.3±0.2 nM) nor the basal level of adenylate cyclase activity (2.8±1.1 pmol cAMP/mg of protein/min) was altered by diabetes mellitus. The dose-response curve for the inhibition of adenylate cyclase byN ⁶-R-phenylisopropyladenosine (R-PIA), however, did show a rightward shift, indicating that diabetic adipocyte membranes were less sensitive to the effects of adenosine than nondiabetic adipocyte membranes. In contrast, the A₁ adenosine receptor-binding characteristics and adenylate cyclase dose-response curve for cerebral cortical tissue were unchanged by diabetes. These findings suggest that diabetes has tissue-specific effects on the A₁ adenosine receptor system. Furthermore, the decreased sensitivity to adenosine potentially worsens the hyperlipidemia associated with diabetes mellitus. Such alterations in the adenosine receptor system may play a previously undescribed role in the pathophysiology of diabetes mellitus and may help explain why some organs are severely affected by diabetes, but others are relatively spared. Understanding these alterations in adenosine receptor function may lead tonovel therapies of this common metabolic disease.     

Serotonin (5-HT) release in the dorsal raphé and ventral hippocampus: Raphé control of somatodendritic and terminal 5-HT release

Summary Somatodendritic and terminal release of serotonin (5-HT) was investigated by simultaneously measuring extracellular concentrations of 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in the dorsal raphé and ventral hippocampus in freely moving rats. Perfusion of tetrodotoxin (TTX, 1M and 10M) into the dorsal raphé simultaneously decreased dorsal raphé and hippocampal 5-HT release. However, following TTX perfusion into the hippocampus (10M), hippocampal 5-HT release was profoundly reduced but dorsal raphé 5-HT remained unchanged.Systemic injections with the 5-HT_1a agonist, buspirone (1.0–5.0mg/kg, i.p.) decreased 5-HT and 5-HIAA and increased HVA concentrations in the dorsal raphé and in the hippocampus. The decreases in raphé and hippocampal 5-HT induced by systemic buspirone were antagonized in rats pretreated with 1.OmM (–) pindolol, locally perfused into the dorsal raphé. Local dorsal raphé perfusion of (–) pindolol alone (0.01–1.0mM) increased dorsal raphé 5-HT and concomitantly induced a small increase in hippocampal 5-HT. Buspirone perfusion into the dorsal raphé did not change (10 nM, 100nM), or produced a small increase (1.0mM) in raphé 5-HT, without changing hippocampal 5-HT.These data provide evidence that 5-HT release in the dorsal raphé is dependent on the opening of fast activated sodium channels and that dorsal raphé 5-HT_1a receptors control somatodendritic and hippocampal 5-HT release.     

Promotion of cell growth by stimulation of cloned human 5-HT1D receptor sites in transfected C6-glial cells is highly sensitive to intrinsic activity at 5-HT1D receptors

5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotransmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimulation of cloned human 5-HT_1D receptor sites by sumatriptan in stably transfected rat C6-glial/5-HT_1D cells promotes cell growth (Pauwels et al. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353:144–156). In the present study, the pharmacology of this growth response was investigated using a broad series of 5-HT receptor ligands. The data were compared with the responses obtained by measuring inhibition of forskolin-stimulated cAMP formation. 5-HT (EC₅₀: 25 nM) promoted cell growth of C6-glial/5-HT_1D cells, and this in contrast to the absence of any measurable effect in pcDNA3-plasmid transfected and non-transfected C6-glial cells. The 5-HT effect could be mimicked by the following compounds (EC₅₀ in nM): zolmitriptan (0.41), 2-methyl-4-(5-methyl[1,2,4] oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127,935; 0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-y)]ethylamine (MK-462; 3.0), and R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (R(+)-8-OH-DPAT; 30.7). These EC₅₀-values correspond to the compounds binding affinities at the human 5-HT_1D receptor site and, with the exception of GR 127,935 and metergoline, also to the EC₅₀-values found by measuring over 5 min inhibition of forskolin (100 M)-stimulated cAMP formation. Prolonged exposure of GR 127,935 (3 h) and metergoline (30 min) to cells yielded EC₅₀ values in the cAMP assay more close to those measured in the mitogenic response. The growth response to sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the apparently silent antagonists methiothepin, ritanserin and ketanserin with potencies similar to blockade of inhibition of stimulated CAMP formation. The 8-OH-DPAT effect also is likely mediated by 5-HT_1D receptors; stereoselectivity was found with its enantiomers at this receptor site and the effect was blocked by ketanserin (1 M) but not by spiperone (1 M). Micromolar concentrations of the 5-HT_1B receptor agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo[3, 2-b]pyril-5-one (CP 93,129) and of the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induced cell growth with a potency that accorded with the affinity of these compounds for the human 5-HT_1D receptor site. These effects were sensitive to ketanserin (1 M) antagonism, but not to blockade by -adrenergic blockers and the 5-HT₂ receptor antagonist 2-anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 501-C-67). The findings suggest that 5-HT_1A, 5-HT_1B and 5-HT₂ receptors are not implicated in 5-HT-stimulated C6-glial/5-HT_1D cell growth. In conclusion, human 5-HT_1D receptors are involved in the growth of C6-glial/5-HT_1D cells. This cellular response is highly sensitive to the intrinsic activity of compounds at 5-HT_1D receptors.     

Pharmacological and molecular discrimination of brain I2-imidazoline receptor subtypes

I₂-imidazoline receptors labelled with [³H]-idazoxan in the rabbit and rat brains displayed high and low affinity, respectively, for the guanidide amiloride; reinforcing the previous definition of I_2A-imidazoline receptors expressed in the rabbit brain and Its-imidazoline receptors expressed in the rat brain. Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both subtypes of I₂-imidazoline receptors. Among the drugs studied, bromoxidine, moxonidine, (+)- and (-)-medetomidine and clorgyline were more potent on the high and/or low affinity sites of 1_2B- than on their corresponding of I_2A-imida-zoline receptors (K _iH ratios 20 to 65). No correlation was found for the potencies of the drugs tested at the low affinity sites of both I₂-imidazoline receptor subtypes. Preincubation (30 min at 25°°C) with 10^-6 M clorgyline reduced by 60% the B _max of [³H]-idazoxan binding to I₂ B-imidazoline receptors in the rat brain, but it did not affect the binding parameters of the radioligand saturation curves to I_2A-imidazoline receptors in the rabbit brain. These results indicated that I_2A- and I_2B-imidazoline receptor subtypes differ in the pharmacological profiles of their high and low affinity sites and in the ability to irreversibly bind clorgyline. In rat cortical membranes western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of 29/30 kDa and two less intense bands of 45 and 66 kDa. In rabbit cortical membranes the antibody used detected proteins of 30, 57 and 66 kDa. It is suggested that different imidazoline receptor proteins (45 vs 57 kDa) may account for the different pharmacological profiles of I2-imidazoline receptor subtypes.     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR MECHANISMS OF ANGIOTENSIN II (AT1a) RECEPTOR ENDOCYTOSIS

• 1 Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT₁; with subtypes AT_1a and AT_1b) and type 2 (AT₂) cell surface angiotensin receptors. Both AT₁ and AT₂ receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT₁ is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors.
• 2 AT₁ endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT₁ receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation.
• 3 The function of AT₁ endocytosis and the reason for the disparity between AT₁ and AT₂ endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types.
• 4 For many receptors that undergo internalization, it has been established that internalization motifs (2–6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin-coated pits and vesicles.
• 5 Mutagenesis studies on the AT_1a receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII-stimulated AT_1a receptor endocytosis.

     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: PHYSIOLOGICAL ACTIONS OF ANGIOTENSIN II MEDIATED BY AT1 AND AT2 RECEPTORS IN THE BRAIN

• 1 Autoradiographic binding studies have shown that the AT₁ receptor is the predominant angiotensin II (AngII) receptor subtype in the central nervous system (CNS). Major sites of AT₁ receptors are the lamina terminalis, hypothalamic paraventricular nucleus, the lateral parabrachial nucleus, rostral and caudal ventrolateral medulla, nucleus of the solitary tract and the intermediolateral cell column of the thoraco-lumbar spinal cord.
• 2 While there are differences between species, AT₂ receptors are found mainly in the cerebellum, inferior olive and locus coeruleus of the rat.
• 3 Circulating AngII acts on AT₁ receptors in the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) to stimulate neurons that may have a role in initiating water drinking.
• 4 Centrally administered AngII may act on AT₁ receptors in the median preoptic nucleus and elsewhere to induce drinking, sodium appetite, a sympathetic vasoconstrictor response and vasopressin secretion.
• 5 Recent evidence shows that centrally administered AT₁ antagonists inhibit dipsogenic, natriuretic, pressor and vasopressin secretory responses to intracerebroventricular infusion of hypertonic saline. This suggests that an angiotensinergic neural pathway has a role in osmoregulatory responses.
• 6 Central angiotensinergic pathways which include neural inputs to the rostral ventrolateral medulla may use AT₁ receptors and play a role in the function of sympathetic pathways maintaining arterial pressure.

     

Effects of β-ODAP and its biosynthetic precursor on the electrophysiological activity of cloned glutamate receptors

3-N-Oxalyl- -2,3-diaminopropanoic acid (β-ODAP) induces neurolathyrism, a motor neuron disease. To elucidate the pathogenic mechanism of this process, the action of β-ODAP on the excitatory amino acid (EAA) receptor-mediated currents was examined using cloned EAA receptors expressed in Xenopus oocytes. On the voltage-clamp recordings of an AMPA receptor (₁/₂ heterooligomer), β-ODAP was a strong agonist on this receptor, the potency being almost the same as -glutamate. On the other hand, β-ODAP had little effect on the glutamate-evoked currents through the expressed NMDA receptor (NR1_A/NR2A), but showed a weak inhibitory effect on the glycine-modulatory site. β-ODAP may cause the neurodegenerative disease, neurolathyrism, mainly through the excitotoxic interaction with AMPA receptors.