Decreased platelet vitamin C in diabetes mellitus: Possible role in hyperaggregation

To determine whether the handling of Vitamin C in the diabetic might be altered and might relate to the increased platelet sensitivity, we have investigated levels of platelet Vitamin C in the diabetic and determined the effects of Vitamin C or on platelet aggregation.

Levels of ascorbic acid, as tested by a lingual method, were significantly lower in diabetics than in normals (p < . 01). Ascorbic acid levels in washed platelets from diabetics were significantly lower than from normals (45. 2±3 μg/10¹⁰ platelets vs. 25. 5±2 μg/10¹⁰ platelets, p < . 001). The effects of ascorbic acid on platelet aggregation were studied by adding ascorbic acid in buffered solution (pH 7. 35) prior to aggregating agents. Ascorbic acid (1000 μg/ml) in platelet-rich plasma consistently inhibited platelet aggregation with threshold concentrations of ADP, epinephrine, and collagen, but enhanced aggregation with arachidonic acid. With washed platelets, ascorbic acid inhibited arachidonic acid-induced aggregation. To rule out an interaction of ascorbi acid and arachidonic acid in the medium, platelets were incubated at 37°C for 10 minutes with varying concentrations of ascorbic acid, rewashed, and aggregation with arachidonic acid tested. Aggregation was inhibited in a linear dose-dependent fashion. Oral ingestion of ascorbic acid (2 gm/day) for seven days by normal non-smoking males produced a marked inhibition of aggregation. In a similar study, platelets from an insulin-dependent diabetic showed no change in aggregation. These results suggest that platelet levels of ascorbic acid may relate to the hyper-aggregation of platelets from diabetics.     

Measurement of CBF and CMRO2 using the continuous inhalation of CO2 and O2: Experimental validation using CO2 reactivity in the anaesthetised dog

Experimental support for the steady state ¹⁵O inhalation technique, as used to measure cerebral blood flow (CBF) and oxygen utilisation (CMRO₂), was obtained by describing the response of the cerebral vasculature to variations in arterial P_CO2 in 6 anaesthetised dogs. Measurements were made using a positron emission tomograph (ECAT II) and arterial blood sampling, during the sequential constant inhalation of C¹⁵O₂ and ¹⁵O₂. Values of CBF and CMRO₂ were calculated for a mixture of white and grey matter, using the steady state tracer equations derived by Jones et al. (1976). The mean CMRO₂ was 3.58 ± 0.81 ml O₂ · 100 ml⁻¹ · min⁻¹, whilst the mean CBF and OER (oxygen extraction ratio) values (for an arterial P_CO2 of 40 mm Hg) were 39.9 ml · 100 ml⁻¹ · min⁻¹ and 0.50 ± 0.06, respectively. Arterial P_CO2 was varied between 20 and 150 mm Hg. CBF was found to correlate closely with arterial P_CO2, resulting in a mean slope (specific reactivity) of 1.52 ± 0.38 ml · 100 ml⁻¹ · min⁻¹ · mm Hg⁻¹. Pooling the flow data resulted in a linear relationship between CBF (% change) and arterial P_CO2 in the range 20–70 mm Hg, with a slope (% reactivity) of 3.2% mm Hg⁻¹ (2 P < 0.001). The oxygen extraction ratio (OER) fell with increasing values of arterial P_CO2 resulting in a stable CMRO₂ throughout each study. There was no correlation between CMRO₂ and artificially increased CBF.

These results support and give confidence in the use of the ¹⁵O inhalation technique for measuring CBF, OER and CMRO₂.     

The involvement of calcium in epinephrine or ADP potentiation of human platelet aggregation

The mechanism by which low doses of epinephrine or ADP potentiate primary platelet aggregation was investigated. Aspirin (lmg/ml)-treated human blood platelets were isolated by albumin density gradient centrifugation. Platelet ⁴⁵Ca uptake associated with epinephrine or ADP addition was determined over a 240 sec time course. Pretreatment of the platelets with ADP (0.5μM) significantly increased aggregation in response to epinephrine (0.1μM). This increased aggregation was associated with a substantially greater ⁴⁵Ca uptake than that which occurred in the presence of epinephrine (0.1μM) alone. The potentiated epinephrine response was inhibited by the Ca²⁺ antagonist verapamil (25μM). This inhibition could in turn be reduced by Ca²⁺ (1mM) addition. Pretreatment of platelets with epinephrine (0.1μM) also increased aggregation in response to ADP (0.5μM). Although this potentiated response was not associated with measurable ⁴⁵Ca uptake, it was nevertheless completely abolished by verapamil (25μM) treatment. These findings suggest that low doses of ADP promote the ability of epinephrine to stimulate an increase in membrane permeability to Ca²⁺.     

Characterization of electrogenic Na/K pump in rat neostriatal neurons

The electrogenic Na/K pump current (Ip) was studied in the dissociated neostriatal neurons of the rat by using the nystatin-perforated patch recording mode. The Ip was activated by external K⁺ in a concentration-dependent manner with an EC₅₀ of 0.7 mM at a holding potential (V_H) of −40 mV. Other monovalent cations also caused Ip and the order of potency was Tl⁺>K⁺, Rb⁺>NH₄⁺, Cs⁺>>>Li⁺. The Ip decreased with membrane hyperpolarization in an external solution containing 150 mM Na⁺, while the Ip did not show such voltage dependency without external Na⁺. Ouabain showed a steady-state inhibition of Ip in a concentration- and temperature-dependent manner at a V_H of −40 mV. The IC₅₀ values at 20 and 30°C were 7.1×10⁻⁶ and 1.3×10⁻⁶ M, respectively. The decay of Ip after adding ouabain well fitted with a single exponential function. At a V_H of −40 Mv, the association (k₊₁) and dissociation (k₋₁) rate constants estimated from the time constant of the current decay at 20°C were 4.0×10² s⁻¹ M⁻¹ and 6.3×10⁻³ s⁻¹, respectively. At 30°C, k₊₁ increased to 2.8×10³ s⁻¹ M⁻¹ while k₋₁ showed no such change with a value of 1.8×10⁻³ s⁻¹. A continuous Na⁺ influx was demonstrated by both the Na⁺-dependent leakage current and tetrodotoxin-sensitive Na⁺ current, which resulted in the continuous activation of the Na/K pump. It was thus concluded that the Na/K pump activity was well-maintained in the dissociated rat neostriatal neurons with distinct functional properties and that the activity of the pump was tightly connected with Na⁺ influxes.     

人原代胶质瘤起始细胞的生物学行为初步研究

目的研究人原代胶质瘤起始细胞（GICs）的表型、自我更新能力、成瘤能力和耐药性等生物行为学特性。方法运用常规培养法和条件培养法对来自手术切除的同一胶质母细胞瘤组织分别进行培养,同时获得贴壁生长的人原代胶质瘤细胞（AGCs）和GICs;通过免疫荧光染色法测定其神经胶质酸性蛋白（GFAP）、CD133和巢蛋白的表达,通过单克隆形成实验和梯度密度成瘤实验检测其成球和成瘤能力,利用细胞计数药盒-8检测两种细胞在嘧啶亚硝脲（ACNU）和长春新碱（VCR）作用下的增殖曲线。结果 GICs呈神经球样生长且表达CD133和巢蛋白;47.8%±5.6%的GICs能够自我更新,而可表达GFAP的AGCs中仅有4.3%±1.6%能形成单克隆;ACNU对GICs的IC₅0=1×10^-3.1mol/L,对AGCs的IC₅0=1×10^-3.9mol/L,两者相比较,相差显著（P<0.05）;VCR对GICs的IC₅0=1×10^-4.3mol/L,对AGCs的IC₅0=1×10^-5.5mol/L,两者相比较,亦相差显著（P<0.05）。结论利用条件培养法可以获得具有明显干性的GICs,与分化的AGCs相比具有更强的自我更新能力和成瘤能力,且GICs对ACNU和VCR较AGCs更为耐药。     

The influence of glutathione and other thiols on human platelet aggregation

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC₅₀=0.61 ± 0.05 mM) was greater than that by cysteine (IC₅₀=13 ± 1 mM) or 6-mercaptopurine (IC₅₀=5.4 ± 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.     

Activation of 5-hydroxytryptamine1A receptors increases the affinity of galanin receptors in di- and telencephalic areas of the rat.

Since galanin in vitro selectively increases theK_D value of 5-HT_1A receptors without altering the binding of 5-HT_1B or 5-HT₂ receptors, we have studied whether 5-HT_1A receptor activation in turn may affect galanin binding in the ventral di- and telencephalon and the substantia nigra of the rat. As analyzed by autoradiography, the binding of¹²⁵I-galanin was increased by about 55% in the presence of 3–30 nM of 8-OH-2-(di-npropylamino)-tetralin (DPAT) in the paraventricular thalamic nucleus, the nucleus reuniens and rhomboideus, the zona incerta, the medial and the lateral hypothalamus, and the medial and the lateral amygdaloid area, but not in the pars compacta of the substantia nigra, which lacks 5-HT_1A binding sites. DPAT (10 nM) reduced the IC₅₀ values of galanin at¹²⁵I-galanin binding sites by approximately 55% within all the analyzed di- and telencephalic regions. The overall increase inB_O values was50 ± 11%. Using the filter wipe technique in cryostat sections at Bregma -2.8 mm covering all the brain regions at this level, DPAT (10 nM) decreased the IC₅₀ values of galanin from21.6 ± 1.1nM (control) to15.5 ± 0.9nM, and increased theB_O values by19.4 ± 4.1%. In membrane preparations from the ventral di- and telencephalon, DPAT decreased the IC₅₀ values of galanin binding sites by20 ± 3% at 100 nM of DPAT. This effect could be completely blocked by the specific 5-HT_1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-pthalimido)butyl]piperazine. GTP (0.1 nM) produced a17 ± 5% increase in the IC₅₀ value of galanin and a23 ± 4% decrease in theB_O value of¹²⁵I_galanin binding sites. However, DPAT (100 nM) was still able to decrease the IC₅₀ values of galanin in the presence of GTP (-8 ± 3%;control-10 ± 3%). TheB_max value of¹²⁵I-galanin binding was not affected by DPAT. The increased affinity of galanin binding sites by DPAT seems to reflect a G-protein-independent intramembrane receptor-receptor interaction between 5-HT_1A and galanin receptors. This interaction may represent an intramembrane inhibitory feed-back mechanism of 5-HT_1A receptor sensitivity, and may be important both under normal conditions and in 5-HT-mediated mental disorders.     

Binding of [125I]insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.

Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.     

Effects of Cathepsin G Pretreatment of Platelets on their Subsequent Responses to Aggregating Agents

Cathepsin G, a proteolytic enzyme from activated leukocytes, can interact with platelets during inflammation and thrombosis. Platelets that have been exposed to cathepsin G in thrombi may recirculate if they are freed during fibrinolysis. To determine whether some of the subsequent functions of such platelets would be impaired, we investigated the responses of cathepsin G-pretreated platelets to agonists that they would encounter in the circulation. Suspensions of washed human platelets were labeled with [¹⁴C]serotonin and resuspended in Tyrode-albumin solution (with 2 mM Ca²⁺ and apyrase). After 15 minute incubation with 400 nM cathepsin G at 37°C, 52±3% of [¹⁴C]serotonin had been released, and glycoprotein Ib was degraded. The platelets were washed and resuspended in fresh medium to remove cathepsin G and released materials. Ristocetin-induced agglutination was abolished, indicating that the binding site for von Willebrand Factor on glycoprotein Ib had been removed. Aggregation and release of residual [¹⁴C]serotonin in response to 0.1–1.0 U/mL thrombin was blocked or greatly reduced by the cathepsin G pretreatment. This inhibition is probably largely due to cleavage by cathepsin G of some of the protease-activated receptors at the C-terminal side of Ser⁴² so that the tethered ligand is lost. Pretreatment with cathepsin G did not affect responses to ADP or a low concentration of platelet-activating factor in the presence of fibrinogen, indicating that receptors for these agonists were unaffected and that the function of the fibrinogen receptor, GPIIb/IIIa was unchanged. Responses to cathepsin G, the thrombin receptor-activating peptide SFLLRN, collagen, or the thromboxane A₂ mimetic U46619 were partially inhibited, even in the presence of added fibrinogen. Platelet adhesion to a collagen-coated surface was 51±7% inhibited, which may indicate cleavage of a collagen receptor or receptors; this may partly account for strong inhibition of collagen-induced aggregation and release of granule contents; additionally, as shown by inhibition of responses to U46619, the function of the thromboxane A₂ receptor may be compromised. Thus, although cathepsin G activates platelets, if they recirculate after interaction with it, their subsequent adhesion to damaged vessel walls, aggregation, and release of granule contents induced by thrombin and collagen will be diminished.     

High- and low-affinity receptors regulate platelet responses to phorbol diesters and teleocidin

We examined binding of ³H-phorbol dibutyrate (³H-PDBu) to gel filtered human platelets (GFP) and discovered that GFP possess two classes of receptors for phorbol diesters (PDE). High-affinity (HA) receptors, approximately 5000/GFP, bound ³H-PDBu with an apparent dissociation constant (K_D) of approximately 12 nM. Low-affinity receptors were approximately 5 times more numerous (2.4 × 10⁴/GFP) and had a 10-fold lower affinity for ³H-PDBu (apparent K_D = 115 nM). The potencies of phorbol myristate acetate (PMA) and PDBu paralleled their binding affinities to the PDE receptors. Teleocidin (Tel), although structurally distinct from PDE, competed with ³H-PDBu for its HA-receptors (K_I Tel = 1.9 nM). Binding of PDE to HA- or LA- receptors was rapid, reversible, saturable and stereospecific. The HA- and LA-receptors modulated different platelet responses. HA-receptors regulated the secretion of β-thromboglobulin from -granules and the release of N-acetyl-β-D-hexosaminidases from lysosomes. LA-receptors mediated both platelet aggregation and the release of serotonin from dense granules. This is the first demonstration of two physiologically active classes of PDE/Tel receptors in human platelets, and demonstrates that particular platelet responses may be directed by distinct classes of receptors for specific agonists.     

H-Clonidine binding to membranes of platelets prepared under physiological conditions

(1) A reproducible binding assay has been established to measure ₂-adrenoreceptors on membranes of human platelets prepared under physiological conditions.

(2) Washed platelet suspensions were obtained from fresh blood by a modified method (Mustard et al., 1972) and membranes were prepared by mechanical homogenization.

(3) ³H-clonidine, a selective ₂-adrenoreceptor agonist was selected as the ligand in a concentration range of 2–64 nM. Specific binding of ³H-clonidine was defined as the difference between total bound radioactivity and that not displaced by excess cold clonidine (6.4 × 10⁻⁵M).

(4) Platelets from 14 healthy young male volunteers were analysed using this technique, and one high affinity binding site, with K_D and B_max values in the reported range was found (K_D: 10.14 ± 1.95 nM; B_max: 428 ± 33 fmol/mg protein (Mean) ± S.E.M.).

(5) This technique will be used in future studies that will examine ₂-adrenoreceptor changes in specific subgroups of depressed patients.     

Mechanisms of vascular graft thrombosis: Role of altered canine platelet sensitivity to thromboxane

Using the standard turbidimetric method of platelet aggregation and quantitation of platelet secretion with ¹⁴C-Serotonin, we have examined the responsiveness of the platelets of mongrel dogs to arachidonic acid (AA), and the thromboxane agonist U46619 in the presence and absence of a subthreshold concentration of epinephrine. In response to stimulation with 750 μM AA, the platelets of 18 dogs produced irreversible aggregation (Group I), the platelets of 22 dogs showed, at most, reversible aggregation (Group II), while the platelets of 8 dogs demonstrated no aggregatory response (Group III). In the presence of AA and a subthreshold concentration of epinephrine (0.5 μM), the platelets of all three groups demonstrated enhanced aggregatory and secretory responses although the extent of ¹⁴C-Serotonin secretion differed significantly between all three groups. These differences in platelet aggregation correlate with the deposition of platelets onto synthetic vascular grafts and the maintenance of graft patency. When stimulated with 0.5 μM U46619 and a subthreshold concentration of epinephrine, the platelets of 97% Group I dogs and 75% of Group II dogs exhibited irreversible aggregation, while the platelets of all Group III dogs showed only reversible aggregation. In addition, significant differences in the extent of ¹⁴C-Serotonin secretion to this combination of agonists were observed between groups. Further examination of the specific effects of U46619 on canine platelets revealed that although the aggregatory and secretory responses to U46619 vary between the different canine platelet populations, the threshold concentration of U46619 required to produce platelet shape change is identical among all groups. Quantitation of the stable metabolite of AA produced via the cyclooxygenase pathway, thromboxane B₂(TxB₂), revealed no significant differences in the production of TxB₂ by the platelets of these different populations upon stimulation with AA. Our results suggest that the mechanisms underlying the differences in responsiveness of canine platelets to AA, are likely due to differences in sensitivity of canine platelets to TxA₂, and may be localized to the mechanism responsible for mediating platelet aggregation and secretion in response to TxA₂.     

Opiate peptide receptors on intact NIE-115 neuroblastoma: radioligand binding properties, intracellular response, and effects of increasing membrane cholesterol

1. Binding of [³H]methionine-enkephalin to intact NIE-115 neuroblastoma cells (competing ligand: naloxone) revealed a homogenous population of receptors with a density (B_max) of 79.0 ± 6.5 mol/mg protein (mean SEM, N=3) and an apparent K, of 5.33 ± 1.63 mM.

2. The order of displacement of [³H]met-enkephalin was met-/leu-enkephalin > naloxone > morphine, suggesting that it is of the delta receptor class.

3. Specific binding was heat-labile, stereospecific and sensitive to Na⁺.

4. Adding met-enkephalin to intact neuroblastoma caused reductions of both basal and prostaglandin E₁-stimulated levels of cyclic AMP (41.4 ± 4.0% (N=6) and 45.1 ± 2.4% (N=3) of control levels, respectively). Maximum inhibition (naloxone-reversible) was observed as low as 10⁻⁷ M met-enkephalin.

5. Preliminary results suggest that cells grown in cholesterol-supplemented medium show reduced binding of [³H]met-enkephalin.     

Epinephrine—Via Activation of P38-Mapk—Abolishes the Effect of Aspirin on Platelet Deposition to Collagen

The mechanism by which epinephrine enhances experimental thrombosis in the presence of aspirin is poorly understood. In this study, we set to explore, in aspirinised platelet-rich plasma (PRP), the effect of epinephrine (100 nmol/l) on platelet deposition to immobilised collagen and the subsequent involvement of several intracellular pathways. Under these experimental conditions, which allow platelet aggregation on top of the collagen-adherent platelets, epinephrine increased platelet deposition by 55–86%. This enhancement could be specifically prohibited by the _2A-adrenoceptor antagonist, atipamezole, the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, and the cytosolic phospholipase A₂ (cPLA₂) inhibitor, mepacrine. The effect of epinephrine coincided with increased phosphorylation of p38MAPK and cPLA₂ and with arachidonic acid (AA) release from platelet membrane. We conclude that epinephrine enhanced platelet deposition on collagen in aspirinised PRP via a mechanism dependent on both free AA in platelet cytosol (released by cPLA₂) and p38MAPK.     

The in vivo effect in humans of pyridoxal-5′-phosphate on platelet function and blood coagulation

Vitamin B₆ has an antithrombotic effect. This, based on the results of in vitro studies, has been attributed to an antiplatelet effect. We assessed the in vivo effect of vitamin B₆ by measuring the effect of long-term administration of vitamin B₆ on platelet function and blood coagulation. Vitamin B₆ (pyridoxine hydrochloride), 100mg twice daily p.o. for fifteen days, was administered to 10 healthy volunteers. The bleeding time was measured before the first dose and 15 days after. A baseline value, the acute effect, chronic effect, and the acute-on-chronic effect of vitamin B₆ was estimated by measuring platelet function. The following tests were performed: platelet aggregation induced by collagen, ADP and epinephrine; thromboxane A₂ (TxA₂)-production and prostacyclin inhibition of ADP-induced aggregation. The effects on the coagulation system were monitored by measuring: the prothrombin time, activated partial thromboplastin time and levels of coagulation factor. Vitamin B₆ significantly prolonged the bleeding time from 4.1 ± 1.1 minutes to 6.8 ± 1.0 minutes (p = 0.0063). Aggregation of platelets with collagen was slightly but not significantly inhibited. Platelet aggregation induced with the agonists ADP or epinephrine was significantly inhibited by vitamin B₆, and the platelets tended to aggregate at a slightly decreased rate. The mean TxA₂-production was slightly, but not significantly, decreased. Vitamin B₆ had no effect on the sensitivity of platelets to prostacyclin, or on the coagulation system. Our results indicate that the antithrombotic effects of vitamin B₆ is limited to inhibition of platelet function; there was no measurable influence on coagulation. The results of this in vivo study are however such that clinical trials are warranted to further assess the efficacy of vitamin B₆ as an antiplatelet drug.     

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

Differential alterations of ion channel binding sites in temporal and occipital regions of the cerebral cortex in Alzheimer''s disease

Three ion channel binding sites were examined by means of quantitative ligand binding autoradiography in temporal and occipital cortex from 9 patients with neuropathologically confirmed Alzheimer's disease (AD) and 7 matched control subjects. The following ligands were used: ¹²⁵I-apamin to label a population of Ca²⁺-sensitive K⁺ channels; [³H]PN200-110 to label L-type voltage-sensitive Ca²⁺ channels and [³H]glibenclamide to label ATP-sensitive K⁺ channels. Ion channel binding sites were compared to: choline acetyltransferase (ChAT) activity and plaque densities measured in the same tissue. In the temporal cortex in AD¹²⁵I-apamin binding was increased compared to controls (e.g. superficial layers: control= 0.71 ± 0.07;AD= 1.02 ± 0.07,mean±S.E.M. pmol/g tissue). In contrast, in adjacent sections [³H]glibenclamide binding was reduced in AD compared to controls (e.g. superficial layers: control= 25.3 ± 1.7;AD= 17.9 ± 1.4pmol/g tissue). [³H]PN200-110 binding in temporal cortex was not altered in AD compared to controls. In the occipital cortex¹²⁵I-apamin binding was increased in AD while both [³H]glibenclamide and [³H]PN-200-110 binding sites in this cortical area were not different from controls. Plaque density (per mm²) was higher in temporal (e.g. layers I–III, 43 ± 6) than in occipital cortex (layers I–III, 27 ± 4) in the AD patients while ChAT was reduced by 40% in temporal cortex and by 50% in occipital cortex compared to controls. The results suggests that the three ion channel binding sites are located on structural elements in the brain which are differentially affected by the pathophysiology of AD.     

Influence of the thromboxane antagonist BM 13.177 on the irrachidonic acid-induced increase in pulmonary vascular resistance and permeability in rabbit lungs

In blood-free perfused isolated rabbit lungs increased availability of free arachidonic acid (AA), whether exogenously applied or released from the endogenous membrane phospholipid pool after different stimuli, causes an acute pulmonary artery pressor response and an increase in vascular permeability. Previous experiments suggested that the vasoconstriction is caused primarily by the cyclooxygenase product thromboxane (Tx) A₂, whereas an increase in the capillary filtration coefficient must be ascribed to non-cyclooxygenase produts of AA. The influence of BM 13.177, a non-prostanoic antagonist of TxA₂₋ and endoperoxide-effects in platelets, on the AA-induced vascular effects in isolated rabbit lungs was investigated. BM 13.177 dose-dependently inhibited the pressor responses evoked by repetitive direct application of AA (IC₅₀ 10⁻⁶ M) or by repetitive stimulation of endogenous AA-release with the calcium ionophore A 23187 (IC₅₀ 10⁻⁷M), with maximum reduction of the pressor responses to < 15 %. The generation of TxA₂ and of prostaglandin (PG)I₂ evoked by these stimuli was, however, not altered. At a concentration of 10⁻⁵ M BM 13.177 did not influence the capillary filtration coefficient, measured during venous pressure challenge, under baseline conditions and after stimulation with AA in presence of indomethacin. Conclusion: βM 13.177 acts as TxA₂/endoperoxide antagonist with dose-dependent inhibition of AA-induced vasoconstriction in the pulmonary vascular bed.     

Lanthanum and zinc sensitivity of GABAA-activated currents in adult medial septum/diagonal band neurons from ethanol dependent rats

The impact of chronic ethanol treatment, sufficient to induce tolerance and physical dependence, on GABA_A receptor function was studied in acutely isolated neurons from the medial septum/nucleus diagonal band (MS/nDB) of adult rats using whole cell, patch-clamp recordings. In ethanol-naive Controls, GABA (0.3–300 μM) induced concentration-dependent increases in Cl⁻ current with a threshold of 0.3–1 μM, a mean maximal current of 7645 ± 2148 pA at 100–300 μM, an EC₅₀ of 11.3 ± 1.3 μM and a slope of 1.53 ±0.07. GABA-activated currents in neurons from animals receiving two weeks of ethanol liquid diet treatment did not differ significantly on any of these measures. The rate of GABA_A receptor desensitization (t_1/2 = 6.49 ± 1.19 s) estimated as the time required for loss of 50% of peak current during sustained application of 10 μM GABA, as well as the residual steady state current remaining following complete desensitization for controls was unchanged by chronic ethanol. The impact of chronic ethanol treatment on the GABA_A receptor modulation by lanthanum and zinc which act as positive and negative allosteric modulators, respectively, was also evaluated. Test pulses of 3 μM GABA in control neurons showed maximal potentiation by 141 ± 30% at ~ 1000 μM lanthanum with an EC₅₀ of 107 ± 34 μM and a slope of ~ 1. Lanthanum potentiation remained the same following chronic ethanol treatment. Initial estimates based on fitted concentration response curves suggested that maximal inhibition of 3 μM GABA responses by zinc at the level of 70.2 ± 8.5% in control cells was significantly increased by chronic ethanol treatment to 95.3 ± 2.5%, although the IC₅₀ of 60.2 ± 25 μM was not changed. However, this difference was not supported by direct tests of maximal 3–10 mM zinc concentrations. These results suggest that chronic ethanol treatment, sufficient to induce tolerance and physical dependence, probably does not lead to readily detectible changes in GABA_A receptor function in MS/nDB neurons.