The role of the lysine binding sites of human plasminogen in the fibrinogen stimulated rate of active site formation in the streptokinase-plasminogen equimolar complex

The effect of various concentrations of -amino caproic acid (EACH) on the rate of active site formation in the human plasminogen moiety of the streptokinase-plasminogen equimolar complex has been studied in the absence and presence of human fibrinogen fragment D₁(FD₁). In the absence of FD₁, the pseudo first order rate constant (k_obs) for active site development in this complex ranges from 8.4–17.9 × 10⁻³ sec⁻¹ with Glul-plasminogen (Glul-Pg), Lys₇₇-plasminogen (Lys₇₇-Pg), and Val₄₄₂-plasminogen (Val₄₄₂-Pg) at levels of EACA from 0–25 mM. In the presence of 2 μM FD₁, the k_obs for active site formation in the SK·Glu₁-Pg complex, of 60.1 × 10⁻³ sec⁻¹, was not altered significantly as the EACA level was increased to 25 mM. Similarly, in the SK· Lys₇₇-Pg complex, the k_obs for active site formation, of 62.1 × 10⁻³ sec⁻¹, was essentially unchanged as the EACA level was increased to 25 mM. Finally, the k_obs for active site formation in the SK·Val₄₄₂-Pg complex, of 113.6 × 10⁻³ sec⁻¹, was also unaffected at levels of EACA up to 5 mM, with a slight inhibition at 25 mM EACA.

These results show that the stimulation of active site formation in the equimolar SK·Pg complex by fibrinogen fragment D₁ is mediated by sites separate from the lysine binding sites of plasminogen.     

Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling

The ability of astrocytes to sequester MeHG may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10⁻⁵ M) significantly inhibited the initial rate (5 min) of uptake of⁸⁶RbCl, used as a tracer for K⁺.⁸⁶RbCl uptake was also sensitive to the omission of medium Na⁺. MeHg (10⁻⁵ M) also markedly inhibited the initial rate of uptake (1 min) of the Na⁺-dependent uptake of [³H]l-glutamate. A second neurotoxin, MnCl₂ (0–5 × 10⁻⁴ M), did not alter [³H]glutamate or⁸⁶RbCl uptake. MeHg, but not MnCl₂, also stimulated the release of intracellular⁸⁶Rb⁺ in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K⁺ gradient. This would be expected to compromise the ability of astrocytes to control extracellular K⁺ either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [¹⁴C]3-O-methyl-d-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0–5 × 10⁻⁴ M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

Electrically induced release of [H]dopamine from slices obtained from different rat brain cortex regions. Evidence for a widespread dopaminergic innervation of the neocortex

Slices obtained from the deeper layers of the rat dorsal frontal, parietal and occipital brain cortex were incubated in vitro with6.25 × 10⁻⁷ M [³H]dopamine (DA), and subsequently superfused and electrically stimulated, while held on quick transfer electrodes, and changes in the efflux of³H and of the individual amines measured. The separation of the amines, with quantitative recoveries, was performed by chromatography on cation-exchange resins eluted sequentially with water,1 NHCl and 6 M urea in1 N HCl. When no drugs were used, the prestimulation efflux was entirely formed by deaminated metabolites, while following stimulation there was an increase in the efflux of deaminated metabolites, and considerable amounts of [³H]-noradrenaline (NA) now appeared. No DA was present in the pre- or poststimulation medium. Similar results were obtained in all the regions studied. When the slices were incubated with10⁻⁵ M desmethylimipramine (DMI),10⁻⁴ M nialamide and 10⁻⁴ M tropolone, before and during incubation with [³H]DA, it was observed that, prior to stimulation, the efflux was composed of deaminated metabolites, DA and 3-methoxytyramine (MTA), and following the electrical stimulus there was an increased release of DA, NA and deaminated compounds (in order of decreasing release), while no change in that of MTA was evident. The stimulus-induced release of DA was greatest from frontal slices, intermediate from parietal, and lowest from occipital ones. DMI-resistant uptake of [³H]DA also diminished when passing from frontal to occipital. These findings are interpreted as due to the presence of dopaminergic axon terminals in all the regions studied, but with a density that diminishes in a rostrocaudal direction.     

Opposite presynaptic regulations by glutamate through NMDA receptors of dopamine synthesis and release in rat striatal synaptosomes

Purified striatal synaptosomes were superfused continuously with L-[3,5-³H]tyrosine to measure simultaneously the synthesis ([³H]water formed during the conversion of [³H]tyrosine into [³H]DOPA) and the release of [³H]dopamine ([³H]DA). Glutamate (10⁻³ M) and NMDA (10⁻³ M, in the absence of Mg²⁺) stimulated the release of [³H]DA, but they reduced the efflux of [³H]water. This reduction of [³H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L--amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [³H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [³H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [³H]DA synthesis and release were mimicked by ionomycin (10⁻⁶ M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.     

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [³H]ACHC and [³H]GABA into cultured neurons and astrocytes was studied. [³H]ACHC uptake was less efficient than that of GABA in both cell types and K_m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V_max values were 0.321 and 0.405 nmol·min⁻¹·mg⁻¹ cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K_i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K_i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.     

Radioimmunoassay for for the detection of active-site specific thrombin inhibitors in biological fluids. I. Assay characteristics and quantitation of recombinant hirudin

A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudinthrombin interaction, a monoclonal antibody to human -thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r. hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.7×10⁻³ to 2×10⁻² antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured -thrombin (from 2×10⁻⁴ to 1× l0⁻² NIH units/ml) in citrated or defibrinated human plasma.

A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace ¹²⁵I- r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin.

The assay was used to test hirudin clearance after bolus i.v. injections of 0.1mg r.hirudin [Val¹-Val²] into human volunteers. The plasma concentrations and elimination kinetics of r. hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed.     

The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets

The effects of acute insulin-induced hypoglycemia on the cerebral NMDA receptor in the newborn were examined by determining [³H]MK-801 binding as an index of NMDA receptor function in 6 control and 7 hypoglycemic piglets. In hypoglycemic animals, the glucose clamp technique with constant insulin infusion was used to maintain a blood glucose concentration of 1.2 mmol/l for 120 min before obtaining cerebral cortex for further analysis; controls received a saline infusion. Concentrations of glucose, lactate, ATP, and PCr were measured in cortex, and Na⁺,K⁺-ATPase activity was determined in a brain cell membrane preparation. [³H]MK-801 binding was evaluated by: (1) saturation binding assays over the range of 0.5–50 nM [³H]MK-801 in the presence of 100 μM glutamate and glycine; and (2) binding assays at 10 nM [³H]MK-801 in the presence of glutamate and/or glycine at 0, 10, or 100 μM. Blood and brain glucose concentrations were significantly lower in hypoglycemic animals than controls. There was no change in brain ATP with hypoglycemia, but PCr was decreased 80% compared to control (P < 0.05). Na⁺,K⁺-ATPase activity was 13% lower in hypoglycemic animals (P < 0.05). Based on saturation binding data, hypoglycemia had no effect on the number of functional receptors (B_max), but the apparent affinity was significantly increased, as indicated by a decrease in the K_d (dissociation constant) from the control value of 8.1 ± 1.6 nM to 5.5 ± 2.1 nM (P < 0.05). Augmentation of [³H]MK-801 binding by glutamate and glycine alone or in combination was also significantly greater in the hypoglycemic animals. These data suggest that acute hypoglycemia may enhance the excitotoxic effects of glutamate in the newborn.     

The kinetic and structural characterization of the reaction of nafamostat with bovine pancreatic trypsin

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., K_i) and the overall inhibition constants (i.e., K_i^*) were calculated to be 11.5 μM and 0.4±0.14 nM, respectively. The second-order rate constant for the reaction was 4.5±0.19×10⁵ M⁻¹s⁻¹, and the product released following the acylation step, 6-amidino-2-naphthol, showed mixed-type inhibition. The competitive (K_ic) and uncompetitive (K_iu) inhibition constants were 14.7 μM and 19.5 μM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s⁻¹ and 195 s⁻¹. The deacylation step was relatively much slower (3.2±0.19×10⁻⁵ s⁻¹), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 μM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.     

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.     

The neuroleptic-like peptide desenkephalin-γ-endorphin does not antagonize the dopamine receptor agonist-induced inhibition of the release of [H]dopamine from rat nucleus accumbens slices in vitro

In rats, the non-opioid β-endorphin (βE) fragment desenkephalin-γ-endorphin (DEγE, βE_6–17) antagonizes the hypomotility induced by a small dose of dopamine (DA) receptor agonists. It has been suggested that DEγE might act in this respect by a direct or indirect blockade of presynaptically located DA receptors in the nucleus accumbens, thereby causing an increase of DA release. Therefore in the present study the effect of DEγE was examined on DA receptor agonist-induced inhibition of the electrically evoked release of previously accumulated [³H]DA from rat nucleus accumbens slices in vitro. The DA receptor agonists apomorphine, LY 171555 andn,n-di-n-propyl-7-hydroxy-2-aminotetralin (DP-7-AT) inhibited in a concentration-dependent manner the electrically evoked release of [³H]DA. The selective D₂ receptor antagonist (−)-sulpiride blocked the effects of apomorphine, corroborating that the DA receptor involved is of a D₂ type. DEγE was tested at several concentrations (10⁻⁹–10⁻⁶) and under various experimental conditions. DEγE, by itself, did not affect either the electrically stimulated or the basal release of [³H]DA. The inhibiting effect of DA receptor agonists was slightly reduced by DEγE, but this effect was present in some experiments only. It is concluded that DEγE does not function as an antagonist for the DA receptor mediating DA release and that the interaction observed in behavioural experiments between DA agonists and DEγE does not occur at the level of this receptor.     

Sensitization of stress-induced feeding in rats repeatedly exposed to brief restraint: the role of corticosterone

The effects of 5-500 μM concentrations of neutral ammonium salts on the binding of ligands to components of the GABA_A receptor complex were investigated. [³H]Flunitrazepam binding to the benzodiazepine receptor was enhanced by ammonium (10–500 μM), but not sodium tartrate with EC₅₀ = 98 μM and E_max = 31%. Further increasing ammonium tartrate concentrations (500–2500 μM) decreased [³H]flunitrazepam binding to control levels. The ammonium tartrate-induced increase in [³H]flunitrazepam binding was manifested as a 50% decrease in K_d. Furthermore, GABA increased the potency of ammonium tartrate in enhancing [³H]flunitrazepam binding by 63%. [³H]Ro 15-1788 and [³H]Ro 15-4513 binding to the benzodiazepine receptor was not significantly enhanced by ammonium tartrate (E_max ≈ 13%). Ammonium tartrate also increased, then decreased the binding of 500 nM [³H]muscimol to the GABA_A receptor (EC₅₀ = 52 μM, E_max = 30%) in a concentration-dependent manner, but had no effect on [³H]SR 95-531 binding (E_max < 16%). The ammonium tartrate-induced alterations in [³H]muscimol binding were demonstrated in saturation assays as the loss of the high affinity binding site and a 27% increase in the _Bmax of the low affinity binding site. These results indicate that ammonia biphasically enhances, then returns ligand binding to both the GABA and benzodiazepine receptor components of the GABA_A receptor complex to control levels in a barbiturate-like fashion. This suggests that ammonia may enhance GABAergic neurotransmission at concentrations commonly encountered in hepatic failure, an event preceding the suppression of inhibitory neuronal function observed at higher ( > 1 mM) ammonia concentrations. This increase in GABAergic neurotransmission is consistent with the clinical picture of lethargy, ataxia and cognitive deficits associated with liver failure and congenital hyperammonemia.     

The selective affinity labeling of factor Xa by peptides of arginine chloromethyl ketone

The preparation of arginine chloromethyl ketones corresponding to the sequence of prothrombin (-Ile-Glu-Gly-Arg-) hydrolyzed by factor Xa in the prothrombin to thrombin conversion has yielded selective and highly effective affinity labels of bovine factor Xa. The Most effective affinity label, DNS-Glu-Gly-ArgCH₂C1, inactivates factor Xa by 50% in 13 min at 2.0 × 10⁻⁹M. Similar rates of inactivation were obtained for Ile-Glu-Gly-ArgCH₂C1 and Ac-Gly-Gly-ArgCH₂C1 at 2.0 × 10⁻⁸ M and for Glu-Gly-ArgCH₂C1 at 2.5 × 10⁻⁷M. DNS-Glu-Gly-ArgCH₂C1 and Ac-Glu-Gly-ArgCH₂C1 were the most selective reagents inactivating factor X_a 16–22 times more effectively than human plasma kallikrein and at least 50 times more effectively than thrombin and plasmin.     

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.     

Histamine H1-receptors mediate phosphoinositide hydrolysis in astrocyte-enriched primary cultures

Astrocyte-enriched primary cultures of newborn rat brain hemispheres, prelabeled with [³H]inositol, accumulated [³H]inositol phosphate but not [³H]inositol bis-and tris-phosphate, after exposure to histamine for 60 min in the presence of 10 mM LiCl. The response to histamine was not a function of contaminating meningeal fibroblasts since no accumulation of [³H]inositol phosphate was elicited by histamine in meningeal cultures. The stimulation of phosphoinositide hydrolysis by histamine in astrocytes was dose-dependent (EC₅₀ = 1.7 μM, maximal effect = 345% over basal levels) and was mimicked by several H₁-receptor agonists. The use of selectiver receptor antagonists confirmed that the histamine response was the result of activation of H₁-receptors. The histamine-induced [³H]inositol phosphate accumulation was completely abolished by omission of Ca²⁺ from the incubation medium. Astrocyte membranes specifically bound the radiolabeled H₁-antagonist, [³H]mepyramine with an affinity (K_d = 5.9 nM) and a density of binding sites (B_max = 113 fmol/mg protein) similar to rat brain. These results demonstrate the presence of functional histamine H₁-receptors in rat brain astrocytes and suggest a role for histamine as a neuromodulator of astrocyte function.     

Transport, uptake, and metabolism of blood-borne vasopressin by the blood-brain barrier

Transport, binding, and metabolism of [phenylalanyl-3,4,5-³H(N)]arginine vasopressin (AVP) by the blood-brain barrier (BBB) was studied in adult guinea-pigs by means of a novel vascular brain perfusion (VBP)/capillary depletion technique and HPLC. A time-dependent, progressive brain uptake of ³H-radioactivity was measured over the 10 min period of VBP both in brain homogenates and in brain tissue depleted of cerebral microvessels. The unidirectional blood-to-brain transport constant, K_IN, estimated by multiple-time tissue uptake analysis of the homogenate and postcapillary supernatant, indicated that the BBB transfer rat ffor [³H]AVP (K_IN = 2.37±0.25 μl min⁻¹ per gram brain homogenate) was almost 10 times higher than for simultaneously perfused [¹⁴C]sucrose, a cerebrosvascular space marker. In contrast to homogenate and postcapillary supernatant, the [³H]radioactivity determined in the vascular pellet after dextran density centrifugation of the brain homogenate was very low and only somewhat higher than for [¹⁴C]sucrose. HPLC analysis of the perfused brain tissue revealed time-dependent degradation of the blood-borne neuropeptide. The percentage of intact [³H]AVP as determined in the postcapillary supernatant progressively declined during brain perfusion, from 49% at 1 min to 11.9% at 10 min. The major detectable labeled metabolite was [³H]phenylalanine, the labeled amino acid residue of [³H]AVP. The aminopeptidase inhibitor bestatin (0.5 mM), perfused simultaneously with [³H]AVP by the VBP technique, did not alter tissue uptake of [³H]AVP, indicating that there was no significant hydrolysis of peptide by the luminal BBB surface. The results suggest that rapid in vivo metabolism of AVP occurs after BBB transport in the brain parenchyma with no evidence of significant capillary sequestration, or degradation of AVP by the BBB.     

Physiological roles of glycine and γ-aminobutyric acid in dissociated neurons of rat visual cortex

The effects of glycine (Gly) and γ-aminobutyric acid (GABA) on the neurons acutely dissociated from rat visual cortex (VC) were investigated in the whole-cell mode using a conventional patch-clamp technique. GABA and Gly evoked Cl⁻ currents (I_Cl) in a concentration-dependent manner at a holding potential (V_H) of −50 mV. The half maximum effective concentrations (EC₅₀) were 4.64 × 10⁻⁶ M for GABA and 6.67 × 10⁻⁵ M for Gly. Strychnine and bicuculline reversively inhibited both 10⁻⁵ M GABA -and 10⁻⁴ M Gly-induced I_Cl in a concentration-dependent manner. The half maximum inhibitory concentrations (IC₅₀) of strychnine on GABA- and Gly-induced currents were 4.00 × 10⁻⁶ M and 8.26 × 10⁻⁸ M, respectively. The IC₅₀ values of bicuculline on GABA and Gly responses were 1.18 × 10⁻⁶ M and 2.97 × 10⁻⁴, respectively. GABA at 10⁻⁵ M, which is near the EC₅₀ of the GABA response, induced I_Cl in all neurons tested n = 83). However, Gly of 10⁻⁴ M, which is near the the Gly response, induced I_Cl in 34 out of 83 neurons tested (41%). Moreover, the maximum amplitude of the Gly response was about 60% of that of the GABA response. On the other hand, the enhancement of N-methyl-d-aspartate (NMDA, 3 × 10⁻⁴M) response by Gly (10⁻⁶ M) was observed in all neurons (n=36) whether they had the Gly-induced I_Cl or not. In conclusion, it was proved that the VC neurons of immature rat could be classified into two classes, i.e. Gly sensitive and Gly insensitive in respect to activation of Cl⁻ channels. There were no significant correlations between the Gly response and the facilitatory action of Gly on the NMDA response, nor between the Gly and GABA responses. The GABA response was closely correlated to the NMDA response, having a correlation coefficient of 0.86.     

Prostacyclin release from the coronary vascular wall by vasoactive substances

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI₂) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 μM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 μM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 μM acetylcholine, 25 μM noradrenaline, 25 μM isoproterenol, 10 μM adenosine triphosphate (ATP 5 μM adenosine, 1 μM angiotensin II, 25 μM histamine or 1 μM serotonin was infused for 30 sec, no inhibitory activity of platele aggregation was observed. Bradykinin (5 × 10⁻⁹ 5 × 10⁻⁶ M) and purified thrombin (1 × 10⁻⁹ 1 × 10⁻⁷ M) induced a dose-dependent release of PGI₂ which was assayed using a radioimmunoassay for 6-keto-prostaglandin F₁ (6-keto-PGF₁).     

Cobalt ion enhancement of 2-chloro[H]adenosine binding to a novel class of adenosine receptors in brain: antagonism by calcium

We have recently reported the identification of a novel class of micromolar-affinity adenosine binding sites in rat brain membranes using the adenosine agonist 2-chloro[³H]adenosine (Cl[³H]Ado). These binding sites are distinguishable from the A1 and A2 adenosine receptors by a number of pharmacological criteria, and we have designated this new class of binding sites as the A3 adenosine binding sites. In the present study, the effects of a wide range of divalent and trivalent cations on micromolar Cl[³H]Ado binding to brain membranes were examined. Co²⁺, Ni²⁺ and La³⁺ markedly stimulated specific Cl[³H]Ado binding by 45–150% above control when tested at concentrations of 1–10 mM. Ca²⁺ had no significant effect on binding except at high concentrations where it depressed binding slightly. Ca²⁺, however, completely prevented the stimulation of Cl[³H]Ado binding by Co²⁺. These findings further distinguish the A3 class of adenosine binding sites from the previously characterized adenosine receptors and suggest that the A3 binding sites are associated with calcium systems in brain.     

Temperature dependence of carbachol-induced modulation of miniature end-plate potential frequency in rats

In the rat soleus, the frequency of miniature end-plate potentials (MEPP) did not change after application of 10⁻⁵ M of the cholinomimetic drug carbachol between 18 °C and 34 °C but decreased by 40% at physiological temperatures of 37–38 °C. The carbachol-induced decrease in MEPP frequency was not eliminated by 10⁻⁷ to 10⁻⁸ M atropine or 3 × 10⁻⁷ (+)-tubocurarine similarly as had been previously found at frog neuromuscular junction.