Georg A. Petroianu

The effect of In vitro hemodilution with gelatin, dextran, hydroxyethyl starch, or Ringer's solution on Thrombelastograph.

To determine the effects of progressive in vitro hemodilution with various plasma substitutes on whole blood coagulation, blood was obtained from six healthy volunteers. The Thrombelastograph® (TEG; Haemoscope Corp., Morton Grove, IL) variables of reaction time, coagulation time, maximum amplitude, and growth angle were determined. The following plasma substitutes were tested: two gelatin solutions (4% gelatin polysuccinate and 5.5% oxypolygelatin); two dextrans (10% dextran 40 and 6% dextran 60); and five hydroxyethyl starch (HES) preparations (6% HES 70/0.5–0.55, 3% HES 200/0.5, 6% HES 200/0.5, 10% HES 200/0.5, and 6% HES 450/0.7). Ringer’s solution was also tested to assist analyzing the intrinsic effect of colloid molecules on blood coagulation. The dilution ratios of citrated blood volume to plasma substitute volume were 10:2, 10:4, and 10:10. Blood coagulation was affected by plasma substitutes when the dilution ratios of citrated blood volume to colloid solution volume were 10:4 and 10:10. TEG variables did not change significantly after in vitro hemodilution with lactated Ringer’s solution. The tested gelatin solutions showed less intrinsic effect on blood coagulation than other plasma substitutes. All HES preparations showed similar intrinsic effects as 6% dextran 60. The plasma substitute of 10% dextran 40 had the strongest effect on coagulation. Coagulation time was the most markedly affected TEG variable. Blood coagulation may be compromised when the dilution ratio of blood volume to colloid solution volume is >10:4. Whereas gelatin solutions have less intrinsic effect on blood coagulation, 10% dextran 40 has the strongest effect on coagulation. Implications: Blood coagulation may be compromised when the dilution ratio of blood volume to colloid solution volume is >10:4. Whereas gelatin solutions have less intrinsic effect on blood coagulation than hydroxyethyl starch or dextran, 10% dextran 40 has the strongest effect on coagulation.

Decreased gene expression of glial and neuronal glutamate transporters after chronic antipsychotic treatment in rat brain

Post-mortem studies in schizophrenic patients revealed alterations in glutamate transporter gene expression. The question concerning a possible influence of chronic antipsychotic treatment on these gene expressions, however, remained open. In this study, 11 rats per group were treated with either haloperidol (1.5 mg/kg per day) or clozapine (45 mg/kg per day) over a period of 6 months in doses comparable to clinic application. Gene expression of glial glutamate transporter EAAT2 and the predominantly neuronal glutamate transporter EAAT3 were examined by in situ hybridization. Clozapine significantly downregulated EAAT2 in hippocampal CA1 and parietal cortex and EAAT3 in the cingulate cortex compared to the expression upon haloperidol treatment and controls. Additionally, clozapine decreased EAAT2 in temporal, cingulate and frontal cortex and downregulated EAAT3 in cingulate and infralimbic cortex, striatum and hippocampal CA1 and CA2 compared to controls. Haloperidol downregulated EAAT2 in cingulate and frontal cortex and reduced EAAT3 in nucleus accumbens, infralimbic cortex and hippocampal CA2. We hypothesize that glutamate transporter downregulation by neuroleptics increases glutamatergic action at the postsynaptic neuron and thereby may be related to beneficial antipsychotic effects and side effects.

Captopril as a Potential Inhibitor of Lung Tumor Growth and Metastasis

Lung cancer is the most common form of cancer in the world, and 90% of patients die from their disease. The angiotensin converting enzyme (ACE) inhibitors are used widely as antihypertensive agents, and it has been suggested that they decrease the risk of some cancers, although available data are conflicting. Accordingly, we investigated the anticancer activity of the ACE inhibitor, captopril, in athymic mice injected with highly tumorigenic LNM35 human lung cells as xenografts. Using this model, we demonstrated that daily IP administration of captopril (2.8 mg/mouse) for 3 weeks resulted in a remarkable reduction of tumor growth (58%, P < 0.01) and lymph node metastasis (50%, P= 0.088). There were no undesirable effects of captopril treatment on animal behavior and body weight. In order to determine the mechanism by which captopril inhibited tumor growth, we investigated the impact of this drug on cell proliferation, apoptosis, and angiogenesis. Immunohistochemical analysis demonstrated that captopril treatment significantly reduced the number of proliferating cells (Ki‐67) in the tumor samples but was not associated with inhibition of tumor angiogenesis (CD31). Using cell viability and fluorescent activated cell sorting analysis tests, we demonstrated that captopril inhibited the viability of LNM35 cells by inducing apoptosis, providing insight about the mechanisms underlying its antitumorigenic activities. In view of these experimental findings, we conclude that captopril could be a promising option for the treatment of lung cancer.

The Nonpsychoactive Cannabinoid Cannabidiol Inhibits 5-Hydroxytryptamine3A Receptor-Mediated Currents in Xenopus laevis Oocytes

The effect of the plant-derived nonpsychotropic cannabinoid, cannabidiol (CBD), on the function of hydroxytryptamine (5-HT)3A receptors expressed in Xenopus laevis oocytes was investigated using two-electrode voltage-clamp techniques. CBD reversibly inhibited 5-HT (1 μM)-evoked currents in a concentration-dependent manner (IC50 = 0.6 μM). CBD (1 μM) did not alter specific binding of the 5-HT3A antagonist [3H]3-(5-methyl-1H-imidazol-4-yl)-1-(1-methylindol-3-yl)propan-1-one (GR65630), in oocytes expressing 5-HT3A receptors. In the presence of 1 μM CBD, the maximal 5-HT-induced currents were also inhibited. The EC50 values were 1.2 and 1.4 μM, in the absence and presence of CBD, indicating that CBD acts as a noncompetitive antagonist of 5-HT3 receptors. Neither intracellular BAPTA injection nor pertussis toxin pretreatment (5 μg/ml) altered the CBD-evoked inhibition of 5-HT-induced currents. CBD inhibition was inversely correlated with 5-HT3A expression levels and mean 5-HT3 receptor current density. Pretreatment with actinomycin D, which inhibits protein transcription, decreased the mean 5-HT3 receptor current density and increased the magnitude of CBD inhibition. These data demonstrate that CBD is an allosteric inhibitor of 5-HT3 receptors expressed in X. laevis oocytes. They further suggest that allosteric inhibition of 5-HT3 receptors by CBD may contribute to its physiological roles in the modulation of nociception and emesis.

On the Interaction of β-Amyloid Peptides and α7-Nicotinic Acetylcholine Receptors in Alzheimer’s Disease

Deterioration of the cortical cholinergic system is a leading neurochemical feature of Alzheimers Disease (AD). This review summarizes evidence that the homomeric α7- nicotinic acetylcholine receptor (nAChR) plays a crucial role in the pathogenesis of this disease, which is characterized by amyloid-β (Aβ) accumulations and neurofibrillary tangles originating from of hyperphosphorylated tau protein. Aβ binds to α7-nAChRs with a high affinity, either activating or inhibiting this receptor in a concentration-dependent manner. There is strong evidence that α7-nAChRs are neuroprotective, reducing Aβ-induced toxicity; but co-localization of α7- nAChRs, Aβ and amyloid plaques also points to neurodegenerative actions. Aβ induces tau phosphorylation via α7-nAChR activation. Aβ influences hippocampus-dependent memory and long-term potentiation in a dose-dependent way: there is evidence that enhancement by picomolar Aβ concentrations is mediated by α7-nAChRs, whereas inhibition by nanomolar concentrations is independent of nAChRs and probably mediated by small Aβ42 oligomers. α7-nAChRs located on vascular smooth muscle cells and astrocytes are also involved in the pathogenesis of AD. Although these data strongly point to an important role of α7-nAChRs in the development of AD, dose-dependence of the effects, rapid desensitization of the receptor and dependence of the effects on Aβ aggregation (monomers, oligomers, fibrils) make it difficult to develop simple therapeutic strategies acting upon this receptor.

Association between myelin basic protein expression and left entorhinal cortex pre-alpha cell layer disorganization in schizophrenia

There is evidence for migrational disturbances in the entorhinal cortex (ERC) in schizophrenia that supports a neurodevelopmental origin of the disorder. Since impaired myelin basic protein (MBP) gene expression during the migration phase could lead to abnormalities in final laminar position, we performed layer specific measurements of MBP expression in the ERC and hypothesised that migrational disturbances of pre-alpha-cell clusters relate to decreased MBP expression. Paraffin embedded sections of the left entorhinal cortex of 16 schizophrenia patients and 10 control subjects were stained for MBP using routine immunohistochemistry. On each section representative regions of interest were scanned to attain optimal quality images of the gray matter. Results were correlated to previous published disturbed dispersion of pre-alpha-cell clusters in adjacent brain sections. Mean MBP stain-intensity was significantly reduced in schizophrenia patients. Absolute MBP stain-intensity was significantly reduced in layers III and IV in patients. A significant correlation of MBP stain-intensity with the distance of the deep pole of the pre-alpha-cell cluster from the gray-white matter junction was observed in the ERC of schizophrenia patients. The present data provide evidence for reduced MBP expression in the ERC in schizophrenia, which implies deficits in axonal myelination and disturbed connectivity. MBP gene is expressed in oligodendrocytes and neuronal populations during embryonic development, which are important in establishing the structure of the cerebral cortex. Correlation between reduced MBP as a sign of down-regulated MBP gene expression and disorganization of pre-alpha-cell clusters supports a neurodevelopmental origin of pathological processes in schizophrenia.

Pretreatment for acute exposure to diisopropylfluorophosphate: in vivo efficacy of various acetylcholinesterase inhibitors

Prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors before exposure to organophosphorus compounds (OPCs) can reduce OPC‐induced mortality. Pyridostigmine is the only FDA‐approved substance for such use. The AChE‐inhibitory activity of known AChE inhibitors was quantified in vitro and their in vivo mortality‐reducing efficacy was compared, when given prophylactically before the exposure to the OPC diisopropylfluorophosphate (DFP). The IC50 was measured in vitro for the known AChE inhibitors pyridostigmine, physostigmine, ranitidine, tiapride, tacrine, 7‐methoxytacrine, amiloride, metoclopramide, methylene blue and the experimental oxime K‐27. Their in vivo efficacy, when given as pretreatment, to protect rats from DFP‐induced mortality was quantified by determining the relative risk of death (RR) by Cox analysis, with RR = 1 for animals given only DFP, but no pretreatment. Physostigmine was the strongest in vitro AChE‐inhibitor (IC50 = 0.012 µ m), followed by 7‐methoxytacrine, tacrine, pyridostigmine and methylene blue. Ranitidine (IC50 = 2.5 µ m), metoclopramide and amiloride were in the mid‐range. Tiapride (IC50 = 256 µ m) and K‐27 (IC50 = 414 µ m) only weakly inhibited RBC AChE activity. Best in vivo protection from DFP‐induced mortality was achieved when physostigmine (RR = 0.02) or tacrine (RR = 0.05) was given before DFP exposure, which was significantly superior to the pretreatment with all other tested compounds, except K‐27 (RR = 0.18). The mortality‐reducing effect of pyridostigmine, ranitidine and 7‐methoxytacrine was inferior, but still significant. Tiapride, methylene blue, metoclopramide and amiloride did not significantly improve DFP‐induced mortality. K‐27 may be a more efficacious alternative to pyridostigmine, when passage into the brain precludes administration of physostigmine or tacrine. Copyright

Protective Drugs in Acute Large-Dose Exposure to Organophosphates: A Comparison of Metoclopramide and Tiapride with Pralidoxime in Rats

Weak and reversible inhibitors of cholinesterase(s), when coadministered in excess with a more potent inhibitor such as organophosphates, can act in a protective manner. The benzamide compound, metoclopramide, confers some protection (putatively via this mechanism) for cholinesterases against inhibition by paraoxon both in vitro and in vivo, after chronic small-dose exposure. Tiapride is a related benzamide. In this study, we compared the protection by metoclopramide and tiapride in rats acutely exposed to large doses of paraoxon with the therapeutic “gold standard,” pralidoxime. Group 1 received 1 &mgr;mol paraoxon (approximately 75% lethal dose), Group 2 received 50 &mgr;mol metoclopramide, Group 3 received 50 &mgr;mol tiapride, Group 4 received 50 &mgr;mol pralidoxime, Group 5 received 1 &mgr;mol paraoxon + 50 &mgr;mol metoclopramide, Group 6 1 &mgr;mol paraoxon + 50 &mgr;mol tiapride, and Group 7 1 &mgr;mol paraoxon + 50 &mgr;mol pralidoxime. All substances were administered intraperitoneally. The animals were monitored for 48 h and mortality was recorded at 30 min, 1, 2, 3, 4, 24, and 48 h. Blood was taken for red blood cell acetylcholinesterase measurements at baseline, 30 min, 24, and 48 h. With the exception of Group 7, in which some late mortality was observed, mortality occurred mainly in the first 30 min after paraoxon administration with minimal changes occurring thereafter. Mortality at 30 min was 0% in the metoclopramide, tiapride, and pralidoxime groups and 73 ± 20 (paraoxon), 65 ± 15 (paraoxon + metoclopramide), 38 ± 14 (paraoxon + tiapride), and 13 ± 19 (paraoxon + pralidoxime). Mortality at 48 h was 75 ± 18 (paraoxon), 67 ± 17 (paraoxon + metoclopramide), 42 ± 16 (paraoxon + tiapride), and 27 ± 24 (paraoxon + pralidoxime). Metoclopramide does not significantly influence mortality after acute large-dose paraoxon exposure. Both tiapride and pralidoxime significantly decreased mortality in our model. The protection conferred by tiapride was significantly less than that conferred by pralidoxime at 30 min, but was not significantly different at 24 and 48 h.

Usefulness of administration of non‐organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using paraoxon

Reversible acetylcholinesterase (AChE) inhibitors can protect against the lethal effects of irreversible organophosphorus AChE inhibitors (OPCs), when administered before OPC exposure. We have assessed in vivo the mortality‐reducing efficacy of a group of known AChE inhibitors, when given in equitoxic dosage before exposure to the OPC paraoxon. Protection was quantified in rats by determining the relative risk (RR) of death.

Effects of phenothiazine-class antipsychotics on the function of α7-nicotinic acetylcholine receptors.

The effects of phenothiazine-class antipsychotics (chlorpromazine, fluphenazine, phenothiazine, promazine, thioridazine, and triflupromazine) upon the function of the cloned α₇ subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were tested using the two-electrode voltage-clamp technique. Fluphenazine, thioridazine, triflupromazine, chlorpromazine, and promazine reversibly inhibited acetylcholine (100 μM)-induced currents with IC₅₀ values of 3.8; 5.8; 6.1; 10.6 and 18.3 μM, respectively. Unsubstituted phenothiazine did not have a significant effect up to a concentration of 30 μM. Inhibition was further characterized using fluphenazine, the strongest inhibitor. The effect of fluphenazine was not dependent on the membrane potential. Fluphenazine (10 μM) did not affect the activity of endogenous Ca²⁺-dependent Cl⁻ channels, since the extent of inhibition by fluphenazine was unaltered by intracellular injection of the Ca²⁺ chelator BAPTA and perfusion with Ca²⁺-free bathing solution containing 2 mM Ba²⁺. Inhibition by fluphenazine, but not by chlorpromazine was reversed by increasing acetylcholine concentrations. Furthermore, specific binding of [¹²⁵I] α-bungarotoxin, a radioligand selective for α₇-nicotinic acetylcholine receptor, was inhibited by fluphenazine (10 μM), but not by chlorpromazine in oocyte membranes. In hippocampal slices, epibatidine-evoked [³H] norepinephrine release was also inhibited by fluphenazine (10 μM) and chlorpromazine (10 μM). Our results indicate that phenothiazine-class typical antipsychotics inhibit, with varying potencies, the function of α₇-nicotinic acetylcholine receptor.