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.     

Enhancement of human neutrophil function by platelets: effects of indomethacin

To characterize the influence of presence of platelets on human neutrophil function, neutrophil oxidative burst, chemotaxis, leukotriene B₄ and prostacyclin generation were examined in the presence of physiologic concentration of platelets (40: 1). Presence of platelets significantly (P 4 generation, these inhibitory effects of indomethacin were attenuated in the presence of platelets. Thus presence of platelets enhances neutrophil activity and overcomes the inhibitory effects of indomethacin on neutrophils.     

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.     

Quantitation of serum prostacyclin-binding in thrombotic thrombocytopenic purpura

We quantitated serum PGI₂ binding of 8 normal subjects and two TTP (thrombotic thrombocytopenic purpura) patients by gel filtration and gel partition methods using a stable PGI₂ analogue, iloprost. The dissociation constant (K_D) and the binding capacity (or binding stoichiometry) determined for the normals were 94 ± S.D. 19 μM and 1.8 ± S.D. 0.5 mM (or 2.0 ± .6, iloprost:HSA). Corresponding values for serum samples obtained from TTP patient I were K_D 200 μM, and B_max 2.3 mM in the acute phase, and 75 μM and 1.8 mM respectively in the remission phase. The serum samples from TTP patient II exhibited a higher K_D. Values of 299 μM (acute phase) and 147 μM (remission phase) were obtained. The corresponding binding capacities were 2.1 mM and 1.5 mM. Binding affinity change appears to be the main factor which resulted in the PGI₂ binding defect in TTP.     

NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation

N₂-laser-induced fluorescence in combination with the time and spectral resolution of fluorescent NADH molecules allows on-line measurement of relative NADH concentration with high spatial resolution (diameter of optical fibre 200 μm, λ_exc = 337 nm, λ_det = 460 nm). Energy metabolism was impaired in submerged rat hippocampal slices using the inhibitors amytal, 3-nitropropionate (3-np), sodium cyanide (1 mM each) and the uncoupling agent 2,4-DNP (200 μM). A microprocessor-controlled repeated positioning of the optical fibre in CA1 and CA3 pyramidal cell layers, and CA1 stratum radiatum (CA1_SR). Time-dependently, NADH fluorescence increased reversibly upon perfusion with amytal and cyanide. It was unchanged by perfusion with 3-np for 40 min and rapidly decreased upon perfusion with 2,4-DNP. The CAl/CA3 ratio of NADH fluorescence mildly decreased to 0.92 ± 0.04 (mean ± S.D.) at 10 min (P < 0.05) and 0.89 ± 0.05 at 211 min (P < 0.01) upon perfusion with amytal. The CA1/CA3 ratio increased to 1.56 ± 0.28 at 10 min (P < 0.01) and 1.29 ± 0.35 at 20 min (P < 0.05) upon application of 2,4-DNP. Fluorescence in CA1_SR, was similar to fluorescence in CA1 upon perfusion with 2,4-DNP and similar to CA3 upon perfusion with amytal. We conclude that NADH fluorescence can be measured with high regional selectivity and specificity in hippocampal slices. Selective inhibition of mitochondrial complex I and uncoupling of energy metabolism differentially impair NADH concentration in different hippocampal areas.     

Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK_Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K⁺ (150 mM) and 1 μM intracellular Ca²⁺ conditions, internal application of β-AgTx inhibited the activity of BK_Ca by reducing open probability (P_o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P_o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK_Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.     

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.     

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.     

Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01–10 μM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC₅ of 0.3 μM. In addition, the inhibitory effect induced by DA at a low concentratiion (0.1 μM) was antagonized by sulpiride (1–10 nM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist SKF-83566 (1–5 μM) did not affect the blocking effect by DA 0.1 μM. Based on these findings, we conclude that DA at a low concentration ( 0.1 μM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D₂, but not D₁ dopaminergic receptors, located on the terminals of corticostriatal neurons.     

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.     

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.     

Brain cell membrane Na,K-ATPase activity following severe hypoxic injury in the newborn piglet

This study tests the hypothesis that severe brain hypoxia causes decreased Na⁺,K⁺-ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3) were studied. Hypoxia was induced by lowering the FiO₂ to maintain PCr/P_i ratio at 25% of baseline for 1 h as monitored by ³¹P-NMR spectroscopy. PCr/P_i returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na⁺,K⁺-ATPase activity was determined. Na⁺,K⁺-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na⁺,K⁺-ATPase activity of 58.3 ± 1.3 μM P_i/mg protein/h (mean ± S.E.M.). Na⁺,K⁺-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 ± 1.3, 47.4 ± 3.6, 48.7 ± 2.9 μM P_i/mg protein/h) (P < 0.05 compared to NC). There was no differene in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxiditive phosphorylation (PCr/P_i) by 48 h, there is a permanent decrease in Na⁺,K⁺-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na⁺,K⁺-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/P_i values cannot be used as an index of recovery of cell function.     

Profiles of percent reduction of cytochromes in guinea pig hippocampal brain slices in vitro

Percent reduction profiles of cytochromes (cyt.) aa₃, bandc were investigated in bloodless guinea pig hippocampal brain slices of 400, 600 and 800 μm in thickness ranging in temperature from 22 to 37 °C. The extent of the percent reduction of cytochromes was compared with the generation of orthodromic potentials elicited by the stimulation of the stratum radiatum, and the cessation of the potentials was found to be correlated with the extent of the percent reduction of the cytochromes. In the case of 400 μm slices, they were found to be in normoxia both from the extent of the percent reduction levels of cytochromes and from the generation of orthodromic responses over a range in temperature. In the case of 600 μm slices, those incubated under temperatures of 22 to 32 °C were not in hypoxia from the levels of cytochrome reduction and the production of a field potential. However, slices at 37 °C were in hypoxia because of cyt. c levels approached those of cyt. b and the orthodromic response was suppressed. In 800 μm slices, those at 22–27 °C were in normoxia; however, slices maintained at 32–37 °C were in hypoxia because the levels of cyt. c reduction closely approximated those of cyt. b at 32 °C whereas those of cyt. aa₃, bandc were almost the same as at 37 °C. Moreover, the orthodromic field potential was not evoked.     

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).     

5-HT inhibits lateral entorhinal cortical neurons of the rat in vitro by activation of potassium channel-coupled 5-HT1A receptors

Serotonin (1–40 μM) reduced input resistance by 20.6±6% and hyperpolarized stellate and pyramidal neurons of layers two and three of the lateral entorhinal cortex. 5-Carboxamidotryptamine, a 5-HT₁ agonist, and the selective 5-HT_1A agonist 8-hydroxy-dipropylaminotetralin mimicked the action of serotonin. The reversal potential of 5-HT-mediated hyperpolarizations was sensitive to the extracellular K⁺ concentration, indicating a potassium conductance change. Serotonin treatment suppressed excitatory amino acid-mediated synaptic potentials (by 48%, K_d=6.9 μM) and responses to exogenously applied glutamate (70.1±17% of control, n=7), but did not alter paired-pulse facilitation, indicating a postsynaptic site of action. Intracellular application of QX-314, a blocker of potassium conductance, significantly reduced depression of synaptic potentials by 5-HT agonists. In cells filled with QX-314, responses to exogenously applied glutamate were not reduced by serotonin or 5-carboxamidotryptamine application. These results indicate that the observed conductance increase associated with 5-HT application accounts for most if not all of the observed depressant effects of 5-HT_1A agonists on excitatory amino acid-mediated neurotransmission.     

Sumatriptan modifies cortical free radical release during cortical spreading depression. A novel antimigraine action for sumatriptan?

Increases in concentration of brain NO are proposed to initiate and mediate migraine headache. Triggered by focal depolarisation, spreading depression (SD) represents a suitable mechanism for eliciting widespread release of nitric oxide. The current study examines the effect of sumatriptan, a 5-HT_1B/1D agonist and effective antimigraine therapy, on free radical release (nitric oxide and superoxide) in SD in the simple and complex cortices of the rat and cat. Following initiation of SD, sumatriptan pretreatment (300 μg kg⁻¹ i.v., 15 min prior to SD) modulated all phases of nitric oxide release associated with each SD in both cats and rats. As a result, superoxide levels were observed to significantly (ANOVA, post hoc LSD) increase versus vehicle treated animals (saline 1 ml kg⁻¹ i.v. 15 min prior to SD) during specific phases of each SD depolarisation. Averaged over all SD depolarisations, mean peak SD nitric oxide levels per depolarisation were 0.73±0.23 μM (n=29) in cats, and 0.42±0.09 μM (n=34) in rats. Sumatriptan significantly (Students t-test, P<0.05, two tailed hypothesis, P<0.05) modulated this increase in cortical nitric oxide concentrations to 0.32±0.06 μM (n=25) and 0.22±0.07 μM (n=37) in cats and rats. Sumatriptan appears to decrease the amplitude of nitric oxide release but enhances extracellular superoxide concentrations in both lissencephalic and gyrencephalic cortices during SD.     

Impairment of endothelium-dependent dilatation of the basilar artery during diabetes mellitus

The goal of this study was to determine whether responses of the basilar artery are altered during diabetes mellitus. We measured the diameter of the basilar artery in vivo in non-diabetic and diabetic rats (streptozotocin; 50–60 mg/kg i.p.). Responses of the basilar artery to agonists, which presumably produce dilatation by releasing endothelium-derived relaxing factor (EDRF), were impaired in diabetic rats compared to non-diabetic rats. Acetylcholine (1.0 and 10 μM) dilated the basilar artery by 13 ± 2 and 26 ± 4% (means±S.E.M.), respectively, in non-diabetic rats, but by only 13 ± 1 and 9 ± 2%, respectively, in diabetic rats (P < 0.05). Bradykinin (1.0 and 10 μM) dilated the basilar artery by14 ± 2 and 35 ± 6 % (means±S.E.M.), respectively, in non-diabetic rats, but by only5 ± 1 and 6± 2%, respectively, in diabetic rats (P < 0.05). The response to nitroglycerin was similar in non-diabetic and diabetic rats. Thus, impairment of vasodilatation in diabetic rats in response to acetylcholine and bradykinin is not related to non-specific impaired of vasodilation than impaired dilator responses of the basilar artery in response to acetylcholine and bradykinin in diabetic rats may be related to the activation of the throm☐ane A₂-prostaglandin H₂ receptor. SQ 29548 (a specific throm☐ane A₂-prostaglandin H₂ receptor antagonist) did not alter responses of the basilar artery to acetylcholine and bradykinin. These findings suggest that diabetes mellitus impairs endothelium-dependent dilation of the basilar artery. In addition, the mechanism of impaired responses of the basilar artery during diabetes mellitus does not appear to be related to the activation of the throm☐ane A₂-prostaglandin H₂ receptor.     

Possible contribution of acetylamrinone and its enhancing effects on platelet aggregation under shear stress conditions in the onset of thrombocytopenia in patients treated with amrinone

Background: Thrombocytopenia is recognized as one of the most common complications when the patients with severe heart failure are treated with cardiotropic phosphodiesterase (PDE)-3 inhibitors. To understand the mechanism of the onset of this complication, we focused on the effects of various PDE-3 inhibitors and its stable metabolite of acetylamrinone on platelet aggregation occurring under physiological shear stress conditions. Method: Blood specimens were obtained from eight apparently healthy adult donors. Platelet-rich plasma was separated after anticoagulation by citrate. The effects of PDE-3 inhibitors of amrinone and olprinone, as well as the stable metabolite of the former of acetylamrinone, on platelet aggregation induced by its exposure to a shear rate of 1200 and 10,800 s⁻¹ were determined by optically modified cone-plate viscometer. Results: Both olprinone and amrinone inhibited platelet aggregation at 10,800 s⁻¹ in a dose-dependent manner with the IC₅₀ value of 14±1 and 61±8 μM (mean±S.D.), respectively, while amrinone significantly inhibited platelet aggregation at 1200 s⁻¹ only at highest concentration tested (100 μM). Contrary to the effects shown with PDE-3 inhibitors, acetylamrinone did not inhibit platelet aggregation at all. Moreover, it even enhanced the aggregation at 1200 s⁻¹ when used with 5 μM. Conclusions: Our results demonstrate possible contribution of the enhancing effects of acetylamrinone on platelet aggregation occurring under blood flow conditions, which reduced the platelet count when occurring in real circulation, to the higher incidence of thrombocytopenia in patients treated with amrinone.     

Quantitative microdialysis of neuropeptide Y

The feasibility of using the difference method of quantitative microdialysis to measure neuropeptide Y (NPY) was evaluated in vitro and in vivo. The accuracy of this method was tested in vitro under steady-state conditions for 3 test solutions containing known concentrations of NPY. The estimated concentrations of NPY were 1.2 ± 0.6, 3.7 ± 0.9, and 15.1 ± 0.7 pg/μl (mean ± SEM) in agreement with the actual concentrations of NPY in the test solutions which were 1.1 ± 0.8, 4.6 ± 0.6, and 14.6 ± 0.5 pg/μl (mean ± SEM of solution samples), respectively. The responsiveness of the estimated NPY_ext measure to changes in the external concentration of NPY was also evaluated in vitro. An accurate estimate of NPY_ext was obtained within the first sampling period (within 15 min) after a 2–3-fold increase in the test solution concentration of NPY and within 2–3 sampling periods (15–45 min) in response to a 2–3-fold decrease in the test solution concentration of NPY. In vivo, the estimated basal concentration of NPY in dialysis samples from probes in the medial basal hypothalamus of anesthetized female rats (n = 4) was 4.0 ± 1.6 pg/μl and increased to 9.5 ± 0.3 pg/μl during K⁺ stimulation. Relative recovery was 22% in vivo under steady-state conditions and ranged from 14% to 30% during dynamic conditions. These results demonstrate that the difference method of quantitative microdialysis accurately estimates picomolar concentrations of NPY in vitro, and is sufficiently sensitive to detect basal and increasing concentrations of NPY in vivo.