Evidence for a dual pathway in platelet activating factor-induced aggregation of rat polymorphonuclear leucocytes

The purpose of this study was to determine the role, if any, of Leukotriene B₄ (LTB₄) in Platelet Activating Factor (PAF)-induced aggregation of rat polymorphonuclear leucocytes (PMNs). Exposure of rat PMNs to 10⁻⁷ M PAF resulted in the release of 4.5 ± 0.7 ng/10⁷ cells of LTB₄ measured by radioimmunoassay. However, the maximum aggregation of PMNs achieved by exposure to LTB₄ (10⁻⁷M) was only 50% of that produced by maximally aggregating concentrations of PAF (10⁻⁷M). 5-Lipoxygenase inhibitors, BW755c and Nafazatrom at concentrations that completely abolished LTB₄ synthesis inhibited the aggregation induced by PAF only by 40% and 50% respectively. Furthermore, desensitisation experiments revealed that the aggregatory response of PMNs to PAF was only partially refractory to prior treatment with LTB₄ whereas the aggregatory response to LTB₄ was completely refractory to prior treatment with PAF. These results suggest that PAF-induced aggregation of rat PMNs is in part mediated by LTB₄ and in part directly by an as yet unidentified mechanism.     

Inhibitory activity of the novel Zn[(OPPh2)(SePPh2)N]2 complex towards the Platelet Activating Factor (PAF) and thrombin: Comparison with its isomorphous Co(II) and Ni(II) analogues

The Zn[(OPPh₂)(SePPh₂)N]₂ complex was prepared by a metathetical reaction between a Zn(II) salt and the deprotonated form of the dichalcogenoimidodiphosphinato ligand [(OPPh₂)(SePPh₂)N]⁻. X-ray crystallography revealed a pseudo-tetrahedral MO₂Se₂ coordination sphere, owed to the asymmetric (O,Se) nature of the chelating ligand. A comparison between the structural features of the M[(OPPh₂)(SePPh₂)N]₂ complexes, M = Co, Ni, Zn, shows that the three complexes are isomorphous. The IR and ³¹P NMR properties of Zn[(OPPh₂)(SePPh₂)N]₂ are analyzed in the framework of its crystallographic structure, and are compared with data on similar systems. The above three complexes, along with the (OPPh₂)(SePPh₂)NH ligand and SeO₂, are investigated as inhibitors of the Platelet Activating Factor (PAF) and thrombin activities. The paramagnetic M[(OPPh₂)(SePPh₂)N]₂ complexes, M = Co, Ni, exhibit an approximately 65-fold increased activity, compared to diamagnetic Zn[(OPPh₂)(SePPh₂)N]₂, in both the PAF- and the thrombin-induced aggregation of washed rabbit platelets (WRP). The above three complexes show a comparable significant inhibitory activity in the PAF-induced aggregation of rabbit platelet rich plasma (RPR). Remarkably, SeO₂, a form of Se present in blood, exhibits a very strong and selective inhibitory activity towards the PAF-induced aggregation of WPR. Our studies extend the dataset of metal complexes investigated as inhibitors of PAF and thrombin.     

Biological activity of lipids of pine pollen on platelet aggregation in correlation with the platelet activating factor

Pollen lipids of a pine species were separated by thin layer chromatography systems. The purified neutral and polar lipid classes were examined for their possible platelet aggregation activity and for their effect on Platelet Activating Factor activity. The lipid fraction comigrating on thin layer chromatography with glycerylether standards was shown to have a remarkable inhibition of Platelet Activating Factor activity on washed rabbit platelets in a concentration of 4.5.10(-6) M. At a ten fold higher concentration these lipids also induced platelet aggregation.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Prostaglandin F2α antagonizes thromboxane A2-induced human platelet aggregation

The effects of prostaglandin F_2α on human blood platelet function were investigated. PGF_2α at 15 μM completely blocked platelet aggregation induced by 500 μM arachidonic acid or 3 μM U46619 but had no effect on aggregatin induced by 7.5 μM ADP. A similar specificity of action was not obtained with either PGI₂ or PGE₂. Thus concentrations of PGI₂ (3 nM) or PGE₂ (20 μ M) which inhibited U46619-induced aggregation by 100% also blocked ADP-stimulated aggregation.The inhibitory properties of PGF_2α were not related to increases in platelet cAMP, since direct measurement of intracellular cAMP revealed that 15 μ M PGF_2α produced no substantial change in cAMP levels. This finding was in direct contrast to results obtained using either PGI₂ or PGE₂. Both PGI₂ (3 nM) and PGE₂ (20 μ M) induced significant increases in platelet cAMP levels.The possibility that PGF_2α directly interacts at the platelet TXA₂/PGH₂ receptor was investigated by measuring [³H]PGF_2α binding to isolated platelet membranes. It was found that [³H] PGF_2α binding reached equilibrium within 30 min at room temperature and could be 90% displaced by addition of 1000 fold excess of unlabelled PGF_2α. Furthermore, when 1000 fold excess of either the TXA₂/PGH₂ “mimetic” U46619 or the TXA₂/PGH₂ antagonist 13-azaprostanoic acid was added, specific [³H] PGF_2α binding was displaced by 95% and 85% respectively. In contrast, the same molar excess of 6-keto-PGF_1α, azo analog 1, or TXB₂, caused displacement of only 15%, 20% or 25% of the [³H] PGF_2α binding. Scatchard analysis indicated that [³H] PGF_2α has two binding sites; i.e., a high affinity binding site with an apparent Kd of 50 nM and a low affinity binding site with apparent Kd of 320 nM. These results suggest that the selective inhibition by PGF_2α of AA or U46619-induced aggregation may be mediated through interaction at the platelet TXA₂/PGH₂ receptor.     

Effects of polyoxyethylene detergent (Brij 58) on platelet aggregation,release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4 · 10^?6 M detergent. Efflux of [¹⁴C]serotonin, ⁴⁵Ca²⁺ and labile aorta contracting substance (thromboxane A₂) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4 · 10^?5 M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca²⁺. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [¹⁴C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8 · 10^?4 to 5 · 10^?3 M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregation agents, while higher concentrations produce membrane destabilization and cell lysis.     

Role of lipoxygenase metabolism in platelet function: Effect of aspirin and salicylate

Aspirin inhibits thromboxane A₂ (TxA₂) production whereas its salicylate moiety inhibits 12-hydroxy-eiosatetraenoic acid (12-HETE) production in the platelet. The significance of the latter effect on platelet function is unclear. We examined the effects of aspirin and salicylate on (i) platelet/ collagen adhesion using ³H-adenine-labelled human platelets and collagen- coated discs, (ii) platelet aggregation induced by thrombin, collagen, ADP and arachidonic acid, and (iii) platelet TxA₂ and 12-HETE synthesis as measured by radioimmunoassay and high pressure liquid chromatography respectively. Aspirin (50 μM) decreased platelet aggregation and increased platelet adhesion. The decrease in aggregation was associated with inhibition of TxA₂ production and the increase in adhesion was associated with enhanced 12-HETE production. Salicylate had the opposite effects. Platelet aggregation was increased and platelet adhesion decreased. The increased aggregation was associated with enhanced TxA₂ production and the decrease in aggregation was associated with inhibition of 12-HETE production. These observations suggest that 12-HETE facilitates platelet adhesion which can be altered by salicylate treatment.     

Stimulation of Phospholipase A2 and Secretion of Catecholamines from Brain Synaptosomes by Potassium and A23187

Abstract: Potassium depolarization of rat brain synaptosomes (containing incorporated l-acyl-2-[¹⁴C]arachidonyl-phosphatidylcholine) stimulated endogenous phospholipase A₁ (EC 3.1.1.32) and A₂ (EC 3.1.1.4), as determined by the formation of [¹⁴C]lysophosphatidylcholine, [¹⁴C]arachidonate, and [¹⁴C]prostaglandins, and also stimulated the secretion of [³H]catecholamines. The phospholipase A₂ stimulation, dependent on calcium, was elicited in resting synaptosomes by A23187 and was demonstrated with incorporated 1-acyl-2-[^l4C]oleoyl-phosphatidylcholine but not with incorporated [^I4C]phosphatidylethanolamine or [^l4C]phosphatidylserine. Inhibitors of phospholipase A₂ [p-bromophenacylbromide (10 μM), trifluoperazine (3 μM), and quinacrine (3 μM) reduced the potassium-stimulated [³H]catecholamine release from synaptosomes to 78, 39. and 55%, respectively, of depolarized controls. The addition of lysophosphatidylcholine increased the release of [³H]norepinephrine to levels observed with potassium depolarization, whereas lysophosphatidylethanolamine, lysophosphatidylserine, and sodium dodecyl sulfate were much less effective. Potassium stimulation of synaptosomes increased the endogenous levels of free arachidonic acid and prostaglandins E₂ and F_2α. Indomethacin and aspirin decreased the amounts of prostaglandins formed, allowed the accumulation of free arachidonic acid, and diminished the potassium-stimulated release of [³H]dopamine. p-Bromophenacylbromide inhibited the formation of prostaglandin F_2α. Addition of prostaglandin E₂ inhibited, whereas prostaglandin F_2α enhanced the release of [³H]norepinephrine. These results suggest that calcium influx induced by synaptosomal depolarization activates endogenous phospholipase A₂, with subsequent formation of lysophosphatidylcholine and prostaglandins, both of which may modulate neurosecretion.     

Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

Abstract

Objective

Redox imbalance either inside platelets or in their immediate surroundings prove detrimental to their physiologic functions during haemostasis. This study was therefore aimed to assess the effect of peroxide radicals on platelet functions and underlying signalling mechanisms using asparagine-conjugated diperoxovanadate (DPV-Asn).

Methods

Platelet aggregation, ATP secretion, TxB₂ release, intra-platelet calcium mobilization, protein tyrosine phosphorylation, GPIIbIIIa activation by PAC1 labelling and sCD40L release (enzyme-linked immunosorbent assay) was monitored using various concentrations of DPV-Asn. Cell viability was assessed by Annexin V labelling, MTT assay, LDH leakage and mitochondrial membrane potential by JC-1.

Results

Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A₂ (TxA₂) generation, intra-platelet [Ca²⁺] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor). This was further corroborated by enhanced tyrosine phosphorylation of numerous platelet proteins including PLC-γ2, which apparently played a central role in transducing peroxide signals to regulate [Ca²⁺] influx and phosphorylation of p38 and ERK1/2 MAP kinase.

Discussion

Peroxide radicals critically regulate the thrombo-inflammatory functions of platelets via the PLCγ2-p38-ERK1/2-TxA₂ pathway, which closely resembles the clinical scenario of various pathologies like hyperglycemia and atherosclerosis during which oxidative stress disrupts platelet functions.     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Effects of exogenous phospholipases on brain membrane phospholipid perturbation, (Na+ + K+)-ATPase activity and cellular swelling of brain slices

Rat brain membranes were incubated with bee venom phospholipase A₂ (PLA₂) or phospholipase C (PLC) from Clostridium perfringens. PLA₂ caused a significant increase in free polyunsaturated fatty acids concomitant with membrane phospholipid degradation as monitored by HPLC and by gas chromatography. Equal concentrations of PLC had a much lesser effect than PLA₂. Divergent and differential effects were shown on deacylation and incorporation of [³H]arachidonic acid in membrane phospholipids. The incorporation of [³H]arachidonic acid into various phospholipids was greatly reduced by PLA₂ (0.018 units/ml) whereas PLC at identical concentration was not effective. PLA₂ inhibited (Na⁺ + K⁺)-ATPase but was not effective on p-nitrophenyl-phosphatase activity whereas PLC stimulated both enzymes. PLA₂ induced swelling of cortical brain slices whereas PLC was not effective. Thus, the severity of the perturbation of membrane integrity, and the inhibition of (Na⁺ + K⁺)-ATPase in brain membranes may play an important role in cellular swelling of brain slices induced by PLA₂.     

Interaction of homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) with DNA of Ehrlich ascites tumor cells

A cytostatic, homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) was reduced with NaB³H₄ and [³H]ASE-treated DNA prepared in vitro. We found that: (1) ASE reacts preferentially with purines; (2) ASE decreases the thermal stability of the double helix upon binding to DNA; (3) [³H]ASE binding sites are clustered along the DNA molecules; (4) ASE binding sites probably represent oligo- or polypurine sequences.     

Application of quantitative deuterium NMR to the study of isotope fractionation in the conversion of saccharides to ethanols

³H-PAF-acether (Alkyl-[1′,2′-³H]-2-acetyl-sn-glyceryl-3-phosphorylcholine) was time-dependently transformed by plasma-free rabbit platelets into an unknown product, “PX”, which was distinct from lyso-PAF-acether. This effect was specific for platelets since plasma only converted ³H-PAF-acether into lyso-³H-PAF-acether and platelets were not effective in metabolizing lyso-³H-PAF-acether. Platelet aggregation did not interfere with the formation of “PX”. The latter is a novel platelet metabolite of PAF-acether with as yet unknown biological properties.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

Dose dependent stimulation of hepatic oxygen consumption and alanine conversion to CO2 and glucose by 3,5,3'-triiodo-L-thyronine (T3) in the isolated perfused liver of hypothyroid rats

The effect of increasing concentration of T3 (10 × 10^?12 – 2000 × 10^?6 M) on O₂-consumption and [¹⁴C]-alanine conversion into [¹⁴C]-CO₂ and [¹⁴C]-glucose was investigated in the isolated perfused liver of hypothyroid starved rats. T3 induced within 1 h an increase (i) in the oligomycine-sensitive O₂-consumption (+ 50–85 %), (ii) in the [¹⁴C]-CO₂-production (+55–102 %), and (iii) in the [¹⁴C]-glucose synthesis (+ 40?80 %). These effects were dose dependent and significant at a concentration as low as 10 × 10^?12 M, reported to represent the free hormone concentration in rat serum under in vivo conditions. The results demonstrate a direct and rapid stimulatory action of T3 in the physiological range on hepatic energy metabolism and glucose production. The effects could not be explained by the thyroid hormone induced nuclear activity.     

The Role of GlycineB Binding Site and Glycine Transporter (GlyT1) in the Regulation of [3H]GABA and [3H]Glycine Release in the Rat Brain

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [³H]-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [³H]GABA overflow with an EC₅₀ value of 0.09 mM. The [³H]GABA releasing effect of NMDA was an external Ca²⁺-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [³H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycine_B site, decreased the [³H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [³H]GABA outflow. These data suggest that glycine_B binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [³H]glycine (IC₅₀ 33 and 16 M) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [³H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [³H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [³H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na⁺ for superfusion of hippocampal slices produced an increased [³H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [³H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [³H]glycine efflux even when buffer with low Na⁺ concentration was applied.     

The structures of bis(1H,5H-S-methylisothiocarbonohydrazidium) di-μ-chloro-octachlorodibismuthate(III) tetrahydrate and tris(1H-S-methylisothiocarbonohydrazidium) esachlorobismuthate(III)

The structures of bis(1H⁺,5H⁺-S-methylisothiocarbonohydrazidium) di-μ-chlorooctachlorodibismuthate(III) tetrahydrate: (C₂H₁₀N₄S)₂(Bi₂Cl₁₀)· 4H₂O (compound [I]) and of tris(1H⁺-S-methylisothiocarbonohydrazidium) esachlorobismuthate(III): (C₂H₉N₄S)₃(BiCl_5.67I_0.33) (compound [II]) were determined from single crystal X-ray diffractometer data. Both compounds crystallize as triclinic (P ); crystals [I] with Z = 1 formula unit in a cell of constants: A = 10.621(3), B = 9.989(5), C = 7.439(3) Å, α = 88.31(2), β = 84.51(2), γ = 68.88(2)°, final R = 0.0427 for 2229 unique reflections with I 2σ(I); crystals [II] with Z = 2 and cell dimensions: A = 14.109(4), B = 12.209(9), C = 8.206(7) Å, α = 103.54(3), β = 104.95(2), γ = 81.96(2)°, final R = 0.0411 for 3637 unique reflections (1 2σ(I)). The structure of [I] is built up of diprotonated organic cations, water molecules and dinuclear centrosymmetric [Bi₂Cl₁₀]⁴⁻ anions held together by N-HCl, N-HO, O-HCl hydrogen bonds and Van der Waals interactions. The [Bi₂Cl₁₀]⁴⁻ complex consists of two edge-sharing octahedra in which three pairs of bonds of similar length are observed (Bi-Cl_av = 2.602(5), 2.712(4), 2.855(5) Å). The structure of [II] consists of monoprotonated cations and [BiCl_5.67I_0.33]³⁻ anions held together by a tridimensional network of hydrogen bonds. Each bismuth atom is octahedrally surrounded by six chlorine atoms, one of which is statistically substituted by a iodine atom.