Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain γ-aminobutyric acid_A (GABA_A) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABA_A-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABA_A-receptors would also exhibit a differential response to halothane as assayed by an in vitro³⁶Cl⁻ influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 μmol/1 GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC₅₀ 336±64 μmol/1 (S.E.M.) vs. 605±110 μmol/1, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7±0.4 nmol·mg⁻¹·3⁻¹, vs. DR 4.7±0.5nmol·mg⁻¹·3s⁻). Halothane (500 μmol/1) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC₅₀ for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC₅₀ 1.8±0.4 μmol/1vs.14.7±0.9 μmol/1 without halothane) than in the DR mice (GABA EC₅₀ 4.7±0.6 μmol/1vs.14.7±0.9 μmol/1 without halothane). This effect of halothane was highly significant, both when compared to control, and between the selected lines (P < 0.001). The findings that halothane enhances GABA-gated flux and enhances GABA's channel gating potency support the hypothesis that differential enhancement of agonist-stimulated chloride permeability at GABA_A receptors could be a mechanism underlying the differential obtunding potency of halothane in DS and DR mice. However, at high GABA concentrations halothane decreased maximal chloride flux, more in DS than in DR mice (P < 0.001), which is not consistent with such a mechanism.     

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.     

NKCC1 and KCC2 prevent hyperexcitability in the mouse hippocampus

During postnatal development of the central nervous system (CNS), the response of GABA_A receptors to its agonist undergoes maturation from depolarizing to hyperpolarizing. This switch in polarity is due to the developmental decrease of the intracellular Cl concentration in neurons. Here we show that absence of NKCC1 in P9–P13 CA3 pyramidal neurons, through genetic manipulation or through bumetanide inhibition, results in a significant increase in cell excitability. Furthermore, the pro-convulsant agent 4-aminopyridine induces seizure-like events in NKCC1-null mice but not in wild-type mice. Measurements of muscimol responses in the presence and absence of NKCC1 shows that the Na–K–2Cl cotransporter only marginally affects intracellular Cl⁻ in P9–P13 CA3 principal neurons. However, large increases in intracellular Cl⁻ are observed in CA3 pyramidal neurons following increased hyperexcitability, indicating that P9–P13 CA3 pyramidal neurons lack robust mechanisms to regulate intracellular Cl⁻ during high synaptic activity. This increase in the Cl⁻ concentration is network-driven and activity-dependent, as it is blocked by the non-NMDA glutamate receptor antagonist DNQX. We also show that expression of the outward K–Cl cotransporter, KCC2, prevents the development of hyperexcitability, as a reduction of KCC2 expression by half results in increased susceptibility to seizure under control and 4-AP conditions.     

Changes of K+ -Cl- cotransporter 2 (KCC2) and circuit activity in propofol-induced impairment of long-term potentiation in rat hippocampal slices

Enhancing inhibition via gamma-aminobutyric acid type A (GABA_A) receptors contributes to anesthetic-induced impairment of long-term potentiation (LTP) of excitatory synaptic transmission, which may account for general anesthesia-associated memory impairment (amnesia). The neuron-specific K⁺–Cl⁻ cotransporter 2 (KCC2) is necessary for fast synaptic inhibition via maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of GABA via GABA_A receptors. To explore a possible role of KCC2-dependent inhibition in anesthetic-induced impairment of LTP, we used field excitatory postsynaptic potentials (fEPSP) recording and immunoblotting to study the effect of propofol on LTP maintenance and KCC2 expression in CA1 region of rat hippocampal slices. We found that propofol (30 μM) not only impaired LTP expression but also prevented LTP-accompanied downregulation of KCC2 without affecting the basal transmission of glutamatergic synapses. Moreover, the recurrent inhibition in hippocampal slices was enhanced by propofol. These propofol-induced effects were completely abolished by picrotoxin, a specific GABA_A receptor-chloride channel blocker. Thus, enhancement of GABAergic inhibition and suppression of neuronal excitability may account for the sustained expression of KCC2 and the impairment of LTP by propofol. Together, this study supports a novel role for KCC2 in LTP expression and gives hints to a molecular mechanism, by which anesthetics might cause impairment of LTP.     

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.     

Electrophysiological characterization of sodium channel types in the HCN-1A human cortical cell line

Electrically evoked sodium currents were recorded under whole-cell patch clamp from undifferentiated HCN-1A cells. Peak sodium currents had a half-maximal activation, V_m0.5, of −22.6 ± 1.0 mV with a voltage dependence, K_m, of 7.28 ± 0.39 mV⁻¹. Steady-state inactivation indicated the presence of two types of sodium channel. One type inactivated with V_h0.5 = −93.8 ± 1.2 mV and k_h = −6.8 ± 0.4 mV⁻¹. The second type of sodium channel inactivated w V_h0.5 = −44.6 ± 1.5 mV and k_h = −7.3 ± 0.4 mV⁻¹. The occurrence of each channel type varied from cell to cell and ranged from 0 to 100% of the total sodium current. No variation in the rate of inactivation was seen when the holding potential was adjusted to eliminate the more negative of the two inactivation components. Application of tetrodotoxin (TTX) or saxitoxin (STX) revealed channel types with two different affinities for each toxin. TTX blocked peak sodium conductance with apparent IC50s of 22 nM and 5.3 μM. STX was more potent, with apparent IC₅₀s of 1.6 nM and 1.2 μM. There was no statistical correlation between toxin sensitivity and steady-state inactivation voltage, suggesting that these properties varied independently among sodium channel types.     

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.     

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.     

GABAc receptors

γ-Aminobutyric acid (GABA) is an important neurotransmitter that mediates inhibition in the vertebrate CNS. Until recently, two receptor subtypes were known: bicuculline-sensitive GABA_A and baclofen-sensitive GABA_B receptors. Several lines of evidence now indicate the existence of a third class of GABA receptor, which is distinct pharmacologically from GABA_A and GABA_B receptors and is found predominantly in the vertebrate retina. These novel GABA_C receptors are Cl⁻ pores. They are insensitive to drugs that modulate GABA_A and GABA_B receptors and are activated selectively bycis-4-aminocrotonic acid.     

GABA, acting at both GABAA and GABAB receptors, inhibits the release of cholecystokinin-like material from the rat spinal cord in vitro.

Superfusion of slices of the dorsal zone of the lumbar enlargement of the rat spinal cord with an artificial cerebrospinal fluid allowed the collection of cholecystokinin-like material (CCKLM) whose Ca²⁺-dependent release could be evoked by tissue depolarization with 30 mM K⁺. Studies on the possible influence of GABA and related agonists on this process showed that the amino acid, the GABA_A agonist, muscimol, and the GABA_B agonist, baclofen, inhibited the K⁺-evoked release of CCKLM from the rat spinal cord in a concentration-dependent manner. Maximal inhibition did not exceed −40% with either agonist. Furthermore, the effects of GABA_A and GABA_B receptor stimulation were not additive. Whereas the effects of muscimol (10 μM) and baclofen (1 μM) could be completely antagonized by bicuculline (1 μM) and phaclofen (10 μM), respectively, complete blockade of the inhibition by GABA (1 μM) could only be achieved in the presence of both antagonists. These data indicate that both GABA_A and GABA_B receptors are involved in the negative influence of GABA onto CCK-containing neurones within the dorsal horn of the rat spinal cord. Apparently, these receptors are not located on CCK-containing neurones themselves, since the inhibitory effect of GABA on the K⁺-evoked release of CCKLM could be completely prevented by tetrodotoxin (1 μM). As CCK acts centrally as an endogenous opioid antagonist, such a GABA-inhibitory control of spinal CCK-containing neurones might participate in the analgesic action of the amino acid via the intrathecal route.     

Uptake of leucine and alanine by cultured cerebral capillary endothelial cells

Pure cultures of rat cerebral capillary endothelium have been used to study the A- and L-systems of amino acid transport. Leucine is taken up by a non-concentrative mechanism that can be saturated, and competivively inhibited by phenylalanine. Uptake is rapid, with equilibiration apparent 3–min (al experiments performed at 37 °C). The K_M for transport was 83 μM ± 26(mean ±S.E.M.., n = 3 which is in good agreement with recent vi vivo report using unanesthtissed rats. Alanine was transported by a saturable, concentrative mechanism. Dependence on Na⁺ -ions was demonstrated by lack of specific uptake in Na⁺ -ffree buffer and reduced uptake after preincubation in ouabain — Na⁺, K⁺ -ATPase inhibitor. The K_M was 325 μM ±88 (mean ± S.E.M., n = 3). The finding ac an active A-system transporter in vitro suggests that the cells may have lost the polarity they demonstrate in vivo. The relevance of these findings to transport of nutrients nd drugs across the blood-brain barrier is discussed.     

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.     

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.     

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.     

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.     

Effects of micromolar and nanomolar calcium concentrations on non-synaptic bursting in the hippocampal slice

To determine how [Ca²⁺]₀ affects non-synaptic epileptogenesis in the CA1 area of hippocampal slices, we compared the extracellularly recorded hyperactivity induced by ACSF containing either micromolar (‘low’-Ca²⁺, LC-ACSF) or nanomolar concentrations of Ca²⁺ (‘zero’-Ca²⁺, ZC-ACSF). Both solutions effectively blocked chemical synaptic transmission but spontaneous bursts developed more quickly and consistently in ZC-ACSF and were longer in duration and more frequent than those recorded in LC-ACSF. Antidromically evoked bursts were less epileptiform, i.e., they exhibited fewer population spikes (PSs), in ZC-ACSF. Increasing [Mg²⁺]₀ or decreasing [K⁺]₀ suppressed spontaneous LC-ACSF bursting but only decreased the intensity and frequency of bursting in ZC-ACSF. Either manipulation increased the epileptiform nature of the antidromically evoked field potential, thereby mimicking the effect of increasing [Ca²⁺]₀ from nanomolar to micromolar levels. Bath application of 250–500 μM GABA commonly arrested spontaneous bursting in LC-ACSF. In ZC-ACSF, GABA decreased the burst frequency but paradoxically superimposed high amplitude PSs on each burst. These effects were reversed by the GABA_A receptor antagonists bicuculline methiodide or picrotoxin (50–100 μM). These results indicate that simply lowering [Ca²⁺]₀ from micromolar to nanomolar concentrations increases the burst propensity and intensity of the CA1 population and can dramatically alter responses to pharmacological agents.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

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).     

The release of B beta 1-42 from fibrinogen and fibrin by plasmin

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH₂-terminal end of the A chain, and of Bβ 1–42 (thrombin-increasable fibrinopeptide B immunoreactivity - TIFPB) which reflect plasmin action on the NH₂-terminal end of the Bβ chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Trisbuffered saline at pH 7.4 and at 37°C. With fibrinogen as substrate the K_m was calculated to be 0.87 μM and the V_max 3.75 × 10⁻⁵ M/min/unit of plasmin. With fibrin I monomer as the substrate the K_m was calculated to be 1.25 μM and the V_max 5.5 × 10⁻⁵ M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH₂-terminal end of the Bβ chain.     

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.