L-lactate reduces in vitro the inhibition of butyrylcholinesterase (BChE) by paraoxon (E 600).

Intoxication with the organophosphorus compound paraoxon (POX), an inhibitor of serine hydrolases, is frequent. Oximes are the only enzyme reactivators clinically available. Serendipitous observation led us to the hypothesis that lactate might attenuate some of the POX effects. In vitro effects of lactate on the inhibition of butyrylcholinesterase (BChE) by POX were assessed in plasma of 12 healthy human volunteers. The determinations were repeated using different lactate and different POX concentrations. The BChE activity determinations were performed in the following settings: (i) baseline untreated plasma (BL); (ii) after addition of POX to plasma (pl+POX); (iii) after POX and plasma were incubated and then lactate was added (pl+POX/lact); (iv) after addition of lactate to plasma (pl+lact); (v) after lactate and plasma were incubated and then POX was added (pl+lact/POX); (vi) after lactate and POX were incubated and then added to plasma (lact+POX/pl). In the micro- and millimolar ranges, lactate is able to abolish in vitro the inhibition of BChE by POX in human plasma when added to plasma prior to POX or when incubated with POX prior to addition to plasma. Lactate added to plasma after POX has no protective effect. In a second set of experiments, the effect of lactate on BChE activity was determined. At high millimolar concentrations, lactate itself inhibits BChE to an extent comparable to POX. Lactate is a mixed inhibitor of BChE, being able to interfere with the enzyme-substrate complex (inhibition constant for the enzyme-inhibitor-substrate complex K'I(EIS) = 81 mM) and the enzyme (inhibition constant for the enzyme-inhibitor complex K(I) (EI) = 26 mM).     

Pralidoxime and l-lactate effects in vitro on the inhibition of acetylcholinesterase by paraoxon: pralidoxime does not confer superior protection

Intoxication with the organophosphorus compound paraoxon (POX), an inhibitor of serine hydrolases, is frequent. Although oximes are the only enzyme reactivators presently available, clinical experience with their use was rather disappointing. Recent work has shown that under certain conditions l-lactate is also able to reduce in vitro the POX inhibition of butyrylcholine- and acetylcholineesterase (BChE and AChE). To assess the practical relevance, if any, of these findings, the protective effects of pralidoxime (PRX) and those of lactate had to be compared in the same in vitro model. Effects of PRX on the inhibition of AChE by POX were assessed in vitro in plasma of 12 (six male and six female) healthy human volunteers. The determinations were repeated using different oxime and different POX concentrations. The AChE activity determinations were performed using the following sampler: sample BL-baseline (or untreated plasma); sample a-after addition of POX to plasma (pl + POX); sample b-after POX and plasma were incubated and then oxime was added (pl + POX/PRX); sample c-after addition of oxime to plasma (pl + PRX); sample d-after oxime and plasma were incubated and then POX was added (pl + PRX/POX); sample e-after oxime and POX were incubated and then added to plasma (PRX + POX/pl). Results were corrected for spurious enzyme 'pseudo-activity' due to interaction between PRX and substrate (acetylthiocholine) in the absence of enzyme. In the micro- and millimolar ranges, PRX is able to protect in vitro AChE from inhibition by POX when added to human plasma prior to POX or when incubated with POX prior to addition to plasma. Adding PRX to plasma after POX has no protective effect. The PRX results were compared statistically with historical lactate data (obtained under identical conditions) using the Wilcoxon matched pairs test, with significance assumed for p = 0.01. No difference between PRX and lactate's protective effect on the AChE inhibition by POX was found in the in vitro model used. We therefore conclude that in vivo testing of lactate as a POX protective agent is warranted.     

L-lactate protects in vitro acetylcholinesterase (AChE) from inhibition by paraoxon (E 600)

Intoxication with the organophosphorus compound paraoxon (POX), an inhibitor of serine hydrolases, is frequent. Oximes are the only enzyme reactivators clinically available. Recent work has shown that lactate is able to reduce in vitro the POX effects on butyrylcholinesterase (BChE). Most of the acute clinical symptoms, however, are caused by inhibition of acetylcholinesterase (AChE). Effects of lactate on the inhibition of AChE by POX were assessed in vitro in plasma of 12 (six male, six female) healthy human volunteers. The determinations were repeated using different lactate and different POX concentrations. The AChE activity determinations were performed in the following settings: (BL) baseline (untreated plasma); (a) after addition of POX to plasma (pl + POX); (b) after POX and plasma were incubated and then lactate was added (pl + POX/lact); (c) after addition of lactate to plasma (pl + lact); (d) after lactate and plasma were incubated and then POX was added (pl + lact/POX); (e) after lactate and POX were incubated and then added to plasma (lact + POX/pl). In the micro- and millimolar ranges, lactate is able to protect in vitro AChE from inhibition by POX when added to human plasma prior to POX or when incubated with POX prior to addition to plasma. Lactate added to plasma after POX has no protective effect. In a second set of experiments, the effect of lactate on AChE activity was determined. At high millimolar concentrations, lactate itself inhibits AChE non-competitively (mixed inhibition) to an extent comparable to POX (inhibition constant K(I) = 254 mM).     

Dose-linear pharmacokinetics of oleanolic acid after intravenous and oral administration in rats

The pharmacokinetics of oleanolic acid was evaluated in vitro and in vivo. From Caco-2 cell permeation studies, oleanolic acid was a low permeability compound with no directional effects, suggesting a low in vivo absorption mediated by a passive diffusion. Oleanolic acid was metabolically unstable following incubation with rat liver microsomes in the presence of NADPH. After intravenous injection at doses of 0.5, 1 and 2 mg/kg doses, oleanolic acid showed dose-linear pharmacokinetics as evidenced by unaltered CL (28.6-33.0 ml/min/kg), Vss (437-583 ml/kg), dose-normalized AUC (16.0-17.9 microg min/ml based on 1 mg/kg) and t1/2 (41.9-52.7 min). Following oral administration of oleanolic acid at doses of 10, 25 and 50 mg/kg, Tmax, t1/2, dose-normalized Cmax (66-74 ng/ml based on 25 mg/kg) and dose-normalized AUC (5.4-5.9 microg min/ml based on 25 mg/kg) were comparable between 25 and 50 mg/kg dose, but the plasma concentrations at 10 mg/kg dose were not measurable as they were below the limit of quantitation (2 ng/ml). The absolute oral bioavailability was 0.7% for oral doses of 25 and 50 mg/kg. The extent of urinary excretion was minimal for both i.v. and oral doses. The very low oral bioavailability of oleanolic acid could be due to a poor absorption and extensive metabolic clearance.     

In vivo metoclopramide protection of cholinesterase from paraoxon inhibition: direct comparison with pralidoxime in subchronic low-dose exposure

The benzamide compound metoclopramide (MCP) protects against cholinesterase inhibition by paraoxon (POX) both in vitro and in vivo. This study evaluates MCP-conferred protection of enzyme activity head to head against the therapeutic gold standard pralidoxime (PRX). Six groups of rats were used. All substances were applied i.p. daily for 5 days, followed by a 2-day rest. The 7-day cycle was repeated eight times. Group 1 received 100 nM POX, group 2 received 50 micro M MCP, group 3 received 100 nM POX + 50 micro M MCP, group 4 received 50 micro M PRX, group 5 received 100 nM POX + 50 micro M PRX and group 6 received saline. Red blood cell acetylcholinesterase (RBC-AChE) measurements were performed at baseline and on day 5 of each 7-day cycle. The sums of enzyme activities over time (weekly values expressed as % of baseline of 100%) were compared using the Mann-Whitney rank order test. A Bonferroni correction of 4 for multiple comparisons was applied. Paraoxon significantly reduced enzyme activities when compared with saline (Sigma = 535 +/- 25 vs 902 +/- 42). Metoclopramide conferred statistically significant in vivo protection from inhibition of RBC-AChE by POX (Sigma = 640 +/- 58). The extent of protection was significantly less than that conferred by the gold standard PRX (Sigma = 765 +/- 57). Metoclopramide, in addition to being less effective as an RBC-AChE protective agent, also caused a failure to thrive in the POX+MCP-exposed rats, as evidenced by the changes in body weight.     

Pharmacokinetics of terlipressin after single i.v. doses to healthy volunteers

The pharmacokinetics of triglycyl-lysine-vasopressin (TGLVP) were studied in healthy male volunteers after single i.v. injections of 5, 10 and 20 micrograms/kg b. wt. The half-life of distribution and elimination was 8 and 50 min, respectively. The volume of distribution was 0.7 l/kg b.wt. and the plasma clearance 9 ml/kg b.wt./min. These values are different from those for arginine-vasopressin and lysine-vasopressin (LVP) but confirm to some extent earlier results on TGLVP. No dose-dependent changes of the pharmacokinetics of TGLVP were evident. The LVP formation after TGLVP is described in principle using a combination of pharmacokinetic and pharmacodynamic data. Therapeutically the results in this study suggest a 4-hour interval between injections.     

Disposition of cefotaxime and its metabolite, desacetylcefotaxime, in rat: application of a pharmacokinetic-protein binding model

1. The pharmacokinetics and protein binding of cefotaxime and desacetylcefotaxime were studied in rat. 2. After i.v. dosing of cefotaxime (100 mg/kg) the concentration-time profiles of cefotaxime and its metabolite desacetylcefotaxime followed biphasic decays, giving the kinetic parameters for cefotaxime: VTss and AUC of 127 ml/kg and 8.2 mg/min per ml, respectively. The beta-elimination half-life was 17 min with Cls of 13.1 ml/min per kg. The average association constant (K x 10(3) M-1) and total protein binding site concentration (Pt x 10(-3) M) for cefotaxime were 3.87 and 0.68, respectively, with saturation of plasma protein binding occurring at about 30 micrograms/ml. The average free fraction of cefotaxime in plasma (Fp) was 0.48. 3. The metabolite desacetylcefotaxime had a plasma Cmax of 74.4 micrograms/ml (35 min). The respective elimination half-life and AUC were 53 min and 7.2 mg/min per ml. The binding profile, unlike that of cefotaxime, was non-saturable with a K value of 13.90M-1. The Fp of desacetylcefotaxime was 0.89. 4. The concentration-time behaviour of total and free desacetylcefotaxime (i.v. bolus, 50 mg/kg) declined biexponentially with respective VTss and AUC of 125 ml/kg and 19.4 mg/min per ml (total drug), and 192 ml/kg and 13.9 mg/min per ml (free drug). The beta-phase half-life of total and free drug was about 36 min, whereas CLs (ml/min per kg) were 2.7 (total) and 3.7 (free). The binding characteristics were in good agreement with those of the metabolite produced in vivo, with a K value of 8.58 M-1. The Fp value of desacetylcefotaxime in plasma was 0.73.     

In vitro protection of plasma cholinesterases by metoclopramide from inhibition by paraoxon

Metoclopramide (MCP) is a dopamine receptor antagonist and serotonine receptor agonist widely used as an antiemetic and gastric prokinetic drug. In addition MCP is a reversible inhibitor of cholinesterases from human central nervous system and blood. MCP may have a cholinesterase protective effect against inhibition by organophosphates. The purpose of the study was to quantify "in vitro" by means of the IC(50)-shift the extent of MCP conferred protection, using paraoxon (POX) as an inhibitor. POX is a widely used organophospate responsible for a large number of accidental or suicidal exposures. Cholinesteratic activities (ChE) (with acetyl-thiocholine (A) and butyryl-thiocholine (B) as substrates) in human plasma were measured photometrically in the presence of different POX concentrations and IC(50) was calculated. Determinations were repeated in the presence of increasing MCP concentrations. It appears that the shift induced by the presence of MCP increases with the MCP concentration in a linear manner. In the presence of a clinically easily achievable plasma concentration of 1 micro M MCP the IC(50) of POX for ChE 'shifts' by a factor of approximately 2-3. The protective effect of metoclopramide on cholinesterases could be of practical relevance in the treatment of paraoxon poisoning. We conclude that in vivo testing of MCP as an organophosphate protective agent is warranted.     

The Role of Pre‐junctional D2‐like Receptors Mediating Quinpirole‐Induced Inhibition of the Vasodepressor Sensory CGRPergic Out‐flow in Pithed Rats

Calcitonin gene‐related peptide (CGRP) released from perivascular sensory nerves plays a role in the regulation of vascular tone. Indeed, electrical stimulation of the perivascular sensory out‐flow in pithed rats produces vasodepressor responses, which are mainly mediated by CGRP release. This study investigated the potential role of dopamine D₁‐like and D₂‐like receptors in the inhibition of these vasodepressor responses. For this purpose, male Wistar pithed rats (pre‐treated i.v. with 25 mg/kg gallamine and 2 mg/kg min. hexamethonium) received i.v. continuous infusions of methoxamine (20 μg/kg min.) followed by physiological saline (0.02 ml/min.), the D₁‐like receptor agonist SKF‐38393 (0.1–1 μg/kg min.) or the D₂‐like receptor agonist quinpirole (0.03–10 μg/kg min.). Under these conditions, electrical stimulation (0.56–5.6 Hz; 50 V and 2 ms) of the thoracic spinal cord (T₉–T₁₂) resulted in frequency‐dependent vasodepressor responses which were (i) unchanged during the infusions of saline or SKF‐38393 and (ii) inhibited during the infusions of quinpirole (except at 0.03 μg/kg min.). Moreover, the inhibition induced by 0.1 μg/kg min. quinpirole (which failed to inhibit the vasodepressor responses elicited by i.v. bolus injections of exogenous α‐CGRP; 0.1–1 μg/kg) was (i) unaltered after i.v. treatment with 1 ml/kg of either saline or 5% ascorbic acid and (ii) abolished after 300 μg/kg (i.v.) of the D₂‐like receptor antagonists haloperidol or raclopride. These doses of antagonists (enough to completely block D₂‐like receptors) essentially failed to modify per se the electrically induced vasodepressor responses. In conclusion, our results suggest that quinpirole‐induced inhibition of the vasodepressor sensory CGRPergic out‐flow is mainly mediated by pre‐junctional D₂‐like receptors.     

Protective effect of N-acetylcysteine against arsenic-induced depletion in vivo of carbohydrate

N-acetylcysteine (NAC), a synthetic aminothiol, possesses antioxidative and cytoprotective properties. The present study evaluates the effect of NAC supplementation on arsenic-induced depletion in vivo of carbohydrates. Arsenic (as sodium arsenite) treatment (i.p.) of male Wistar rats (120-140 g b.w.) at a dose of 5.55 mg/kg body weight (35% of LD50) per day for a period of 30 days produced a significant decrease in blood glucose level (hypoglycemia) and a fall in liver glycogen and pyruvic acid contents. The free amino acid nitrogen content of liver increased while that of kidney decreased after arsenic treatment. Arsenic also enhanced the liver lactate dehydrogenase activity whereas glucose 6-phosphatase activity in both liver and kidney decreased significantly following arsenic treatment. Transaminase activities in liver and kidney were not significantly altered except the glutamate-pyruvate transaminase activity that was reduced in kidney after arsenic treatment. Oral administration of NAC (163.2 mg/kg/day) for last 7 days of treatment prevented the arsenic-induced hypoglycemia and glycogenolytic effects to an appreciable extent. There was also recovery of liver pyruvic acid as well as liver and kidney free amino acid nitrogen content after NAC supplementation. Arsenic-induced alteration of glucose 6-phosphatase activity in both liver and kidney was also counteracted by NAC. It is suggested that carbohydrate depletion in vivo due to exposure to arsenic can be counteracted by NAC supplementation.     

Protective effect of apocynin, a NADPH-oxidase inhibitor, against contrast-induced nephropathy in the diabetic rats: a comparison with n-acetylcysteine

The aim of this study was to investigate the effects of apocynin, a NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase inhibitor, in diabetic rats with nephropathy induced by contrast medium (CIN). Diabetes was induced in male Wistar rats by a single dose of streptozotocin (60 mg/kg i.v.). Animals were then divided into the following groups: 1) control group (diabetic rats treated i.v. with saline solution); 2) iomeprol group (iomeprol at 10 ml/kg was injected i.v. 30 min after saline administration); 3) apocynin group (identical to the iomeprol group, except for pre-treatment with apocynin 5mg/kg i.v., 30 min before iomeprol injection) and 4) N-acetylcysteine group (NAC) (same as iomeprol group, except for the treatment with NAC 20 mg/kg i.v. 30 min before iomeprol injection). CIN in animals were assessed 24h after administration of iomeprol. Apocynin significantly attenuates the impaired glomerular function, concentration of Na(+), K(+), alpha glutathione S-transferase levels in urine and neutrophil gelatinase-associated lipocalin levels in plasma caused by iomeprol. In kidney, immunohistochemical analysis of some inflammatory mediators, such as nitrotyrosine, poly-ADP-ribosyl polymerase, tumor necrosis factor-α, interleukin-1β as well as apoptosis (evaluated as terminal deoxynucleotidyltransferase-mediated UTP end labeling assay) revealed positive staining in tissue obtained from iomeprol group. These parameters were markedly reduced in animals treated with apocynin. Similarly, these parameters were also markedly modified by NAC pre-treatment. Here, we have shown that apocynin attenuates the degree of iomeprol-induced nephropathy in diabetic rats.     

Toxicokinetics of methyl paraoxon in the dog

The toxicokinetics of methyl paraoxon, the active metabolite of the organophosphorus insecticide methyl parathion, were studied in non-anaesthetized dogs after intravenous (2.5 mg/kg) and oral (15 mg/kg) administration of methyl paraoxon. After intravenous administration, distribution and elimination occured very rapidly and using the data from 5 min post-injection, the plasma concentration versus time curves could be fitted to a one-compartment open model. The mean half-life of elimination was 9.7 min, the average volume of distribution 1.76 l/kg and the average plasma clearance 126 ml/kg/min. After oral administration, peak plasma concentrations were obtained within 3–16 min, and the bioavailability varied from 5 to 71%. The hepatic extraction of methyl paraoxon measured in anaesthetized dogs, was high (70–92%). Comparison of the urinary excretion after intravenous and oral administration in two dogs indicated a gastrointestinal absorption of more than 60%. The kinetics of methyl paraoxon were linear in the dose range tested.     

Bioavailability of salvianolic acid B in conscious and freely moving rats

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 microl) was vortex-mixed with 50 microl of internal standard solution (chloramphenicol 10 microg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile-methanol-20mM NaH(2)PO(4) (adjusted to pH 3.5 with H(3)PO(4)) (20:10:70 v/v/v) containing 0.1mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-200 microg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC-MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030+/-565 and 582+/-222 min microg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.     

Function of plasma and microsomal enzymes in soman toxicity

Such organophosphorous (OP) nerve agents as sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate) are effective inhibitors of acetylcholinesterases (AChE), butyrylcholinesterases (BChE) and carboxylesterases (CaE). The acute toxicity of these compounds in mammals is known to be mediated through inhibition of AChEs, which leads to increased acetylcholine (ACh) levels. The aim of this study was to compare the significance of the plasma CaEs, microsomal CaEs and CYP450 enzymes in detoxification of soman with and without physostigmine treatment. The mice received physostigmine (0.1 mg/kg body wt) intravenously (i.v.) 10 min prior to the intraperitoneal (i.p.) injection of soman (0.400-0.650 mg/kg body wt in olive oil). To avoid possible signs of poisoning, the animals received atropine sulfate (37.5 mg/kg body wt in saline) subcutaneously (s.c.) immediately after the soman administration. In the present study, the inhibitory effect of soman was greater in plasma CaE than in hepatic microsomal CaE fraction. In addition, soman or the combination of soman-physostigmine had no remarkable effect on the microsomal CaE or P4502B activities. In spite of this, however, the microsomal CaEs might offer more protection against multiple LD50s of soman.     

Acute toxicity of nimbolide and nimbic acid in mice, rats and hamsters

Nimbolide and nimbic acid are toxic to mice only when given i.p. and i.v. but they are less toxic to rats and hamsters. The LD50 values of a single i.p. administration of nimbolide to adult male, female and weanling mice were 225, 280 and 240 mg/kg body wt, respectively, and its i.v. LD50 value was decreased to 24 mg/kg body wt in adult male mice. No fatality was observed when nimbolide was given i.g., i.m. and s.c. to adult male mice. Estimated LD50 values of nimbolide in rats and hamsters were somewhat higher than 600 and 500 mg/kg body wt. After 12-23 h i.p. administration of a lethal dose, most animals died of possible dysfunctions in kidney (tubular necrosis), small intestine (hemorrhagic necrosis), pancreas (acinar cell necrosis) and liver (mild fatty infiltration and focal necrosis). In contrast, mice and rats given a lethal dose of nimbolide (i.v.) died of a marked and sudden drop in arterial blood pressure and respiratory paralysis within about 1-18 min. Nimbic acid was less toxic to mice with an i.v. LD50 value of 265 mg/kg body wt and i.p. and i.g. LD50 values of higher than 600 mg/kg body wt. The possible cause of death induced by nimbic acid may be similar to that of nimbolide given i.v. and this is a sudden hypotensive shock.     

Effect of aminooxyacetic acid (AOAA) on GABA levels in some parts of the rat brain

Summary The accumulation of GABA in the cerebellum and medulla oblongata-pons of rats has been studied after inhibition of GABA-T (EC 2.6.1.19) by different doses of AOAA. It was found that intraperitoneal (i.p.) injections of AOAA were, at least during the first hour after injection, much less effective than intravenous (i.v.) injections probably due to poor absorption i.p. After i.v. injection, AOAA caused a maximal accumulation of GABA in the cerebellum at a dose of 50 mg/kg. This maximal effect was virtually unchanged up to a dose of 150 mg/kg (the highest dose tested i.v.). If GAD (EC 4.1.1.15) was inhibited by 3-mercaptopropionic acid 30 min after AOAA (90 mg/kg i.v.) the GABA level was stable for at least another 30 min. The rate of GABA accumulation in the cerebellum during the first 15 min after AOAA (50–150 mg/kg i.v.) was 0.086 mol/g/min and thereafter 0.034 mol/g/min. It is concluded that AOAA in vivo in a wide dose range inhibits GABA-T almost 100% without affecting GAD to any great extent, and that the onset of action is rapid after i.v. but not after i.p. injection.     

Bioavailability of oral isbufylline in rabbits.

The bioavailability of isbufylline was assessed in male rabbits given 12 mg/kg i.v. (intravenous) or per os (oral) according to a randomized design. The concentrations of unbound (fu = 54.0) isbufylline were considered in plasma as a function of time, after i.v. and oral administration. After oral administration in saline solution, a mean absolute oral bioavailability of 59.6% was calculated. The drug is rapidly absorbed and the comparison of the kinetic profiles after i.v. and per os administration, revealed a rapid elimination: t1/2 27.3 and 28.8 min respectively, and a total body clearance of 67.06 ml/min/kg. Urinary recovery 0-48 h accounted for less than 1% of the dose.     

CK-2289 and milrinone in dogs with propranolol-induced heart failure: Effect of ouabain

We examined the interaction between ouabain and CK-2289, a new bivalent inodilator, and compared their effect with that of milrinone on the hemodynamic and myocardial energetic parameters of anesthetized dogs with propranolol-induced heart failure (PIHF). Mongrel dogs (13–19 kg) of either sex were anesthetized with pentobarbital sodium (35 mg/kg, i. v.) and instrumented for routine hemodynamic measurements using an open-chest, artificially ventilated preparation. PIHF was produced by decreasing left ventricular (LV) dP/dT_max by 50% from control values with an initial infusion of 0.5 mg/kg of propranolol followed by continuous infusion of 0.02 to 0.08 mg/kg of propranolol to maintain PIHF. This was followed by infusion of saline (2 ml, i. v., n = 12/group) or ouabain (25 μg/kg, i. v., n = 12/group). Thirty min later saline and ouabain-treated animals (4/group) were given 2 doses of saline (1 and 2 ml, i. v.), CK-2289 (0.01 and 0.03 mg/kg, i. v.) or milrinone (0.03 and 0.1 mg/kg, i. v.) 30 min apart. Hemodynamic parameters were monitored continuously. Myocardial oxygen consumption (MVO₂) was monitored 15 and 30 min after each dose of drug. CK-2289 increased LV dP/dT_max and LV dP/dT_min by 60 and 120% and 42 and 43%, respectively. Mean arterial pressure decreased by 12% after the high dose of CK-2289. CK-2289 did not affect heart rate, while LV end diastolic pressure decreased by 5 mmHg. CK-2289 increased LV work and did not affect or decrease LV contractile efficiency. Ouabain enhanced the myocardial energetic profile of CK-2289 by allowing CK-2289 to stimulate more work at a lower MVO₂, thereby increasing myocardial efficiency. Milrinone had a profile similar to CK-2289 but in ouabain-pretreated animals with PIHF milrinone stimulated less work at the same MVO₂, thus decreasing contractile efficiency. Thus, ouabain may enhance the myocardial energetic effects of CK-2289.     

Studies on early stage changes of peroxide lipid in isoproterenol-induced myocardial injury]

The amount of peroxide lipid in vivo in the early stage of the experimental model of myocardial infarction in a rat induced by the administration of isoproterenol (Isp) was measured as the value of malonic dialdehyde (MDA). The model of myocardial infarction was made by giving 75 mg/kg of Isp to the rat weighing 270 +/- 10 g. After the administration of Isp, the amounts of lipid in the serum and in the myocardial tissue were measured, and a blood chemistry test (glutamic oxaloacetic dehydrogenase, glutamic pyruvic transaminase, lactate dehydrogenase, free fatty acid, creatine kinase) was simultaneously carried out on the serum. The value of the amount of peroxide lipid in the serum began to elevate 3 h after the administration of Isp and reached a maximum value at 6 h. The value of the amount of peroxide lipid in the tissue began to elevate 30 min after the administration and reached a maximum at 3 h. Each blood chemistry disclosed the elevation 30 min after the administration. As mentioned above, the production of peroxide lipid in vivo on the myocardial disorder in the early stage after the administration of Isp and the biochemical changes showed a significant correlation. From these results it is suggested that the myocardial disorder induced by the administration of Isp has already developed at 30 min after the administration.     

Prevention of tolerance to the organophosphorus anticholinesterase paraoxon with carboxylesterase inhibitors

The contribution of carboxylesterase (CarbE) to the development of tolerance to the organophosphorus anticholinesterase (OP-ANTIChE) paraoxon (diethyl p-nitrophenyl phosphate) was investigated in rats. Daily injections (20 days) of paraoxon (0.09 mg/kg) led to a cumulative dose that was 9.0-fold higher than the acute ED50 of 0.20 mg/kg, s.c. During this period, the rats did not demonstrate visible signs of cholinergic hyperactivity nor did they die, despite the persistence of critically reduced brain acetylcholinesterase (AChE) activity (20-30% of control). In addition, none of these rats died following the administration of a dose of carbachol (3.1 mg/kg, i.p.) that was an LD90 in untreated rats. Daily treatment with the CarbE inhibitors CBDP [2-(o-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide] (2 mg/kg, s.c.) or iso-OMPA (tetraisopropylpyrophosphoramide) (3 mg/kg, i.p.) followed by paraoxon (0.09 mg/kg, s.c.) 60 min later prevented the development of tolerance to paraoxon, since signs of cholinergic hyperactivity were observed and rats died on day 4 of the combined treatment. In tolerant rats, one-time CBDP or iso-OMPA pretreatment increased toxicity to paraoxon, causing the death of all rats within 60 min. The increase in paraoxon toxicity was correlated with inhibition of a plasma CarbE, with high affinity toward alpha-naphthyl acetate (alpha-NA) and to the inhibitors CBDP, iso-OMPA, and paraoxon. Inhibition of a plasma CarbE with high affinity toward p-nitrophenyl acetate (p-NPA) and low affinity to the above inhibitors did not potentiate paraoxon toxicity significantly. Neither the liver CarbEs, which showed high affinity to iso-OMPA, nor the inhibition of butyrylcholinesterase (BuChE) by iso-OMPA in plasma and liver potentiated paraoxon toxicity. By eliminating plasma CarbE (alpha-NA) as potential binding sites for paraoxon with either CBDP or iso-OMPA, paraoxon can exert its toxicity to a greater extent at its specific target site, the functionally important AChE at cholinergic synapses. It is concluded that plasma CarbE (alpha-NA) provided a significant protection against paraoxon intoxication and that the inhibition of this enzyme prevented the tolerance development seen with repeated paraoxon treatments.