New trends in the search for nootropic preparations

The paper describes the effects of the new nootropic agents nooglutyl and glycine N-phenyl-L-prolyl ethyl ester (GVS-111). Nooglutyl, a derivative of L-glutamic and oxynicotinic acids, that has glutamatergic effects is a highly active drug in treating disturbances of memory and learning, protecting against ischemic neuronal damage and brain injury. GVS-111 is a substituted prolyl dipeptide that has the properties of enhancing cognitive functions and is able to prevent the learning impairment provoked by shock, scopolamine, brain injury, and other damages. Multimodal mechanisms responsible for the nootropic effects of nooglutyl and GVS-111.     

The novel substituted acylproline-containing dipeptide, GVS-111, promotes the restoration of learning and memory impaired by bilateral frontal lobectomy in rats

The present study investigated the potential benefit of the ethyl ester of N-phenylacetylprolylglycine (GVS-111) on the model of bilateral frontal lobectomy (BFL) in rats. The animals in Experiment 1 were trained in an active avoidance task and subsequently underwent BFL. The animals in Experiment 2 were first assessed in an open field and in a passive avoidance test before the BFL was performed. BFL dramatically decreased performance in the active avoidance test, disturbed habituation of horizontal activity in the open field and diminished the latency to enter the dark compartment in the passive avoidance test. GVS-111, administered in a dose of 0.5 mg/kg/day i.p. for 9 days following the operation, was found to improve performance in both active avoidance and passive avoidance and restored habituation of horizontal activity in the lobectomized animals.     

Pharmacokinetic analysis of hepatic uptake of galactosylated bovine serum albumin in a perfused rat liver

Hepatic uptake of 111In-labelled galactosylated bovine serum albumin (Gal-BSA) with different number of galactose residues per BSA were studied in rat liver perfusion experiments. During a single-pass constant infusion mode, [111In]Gal-BSAs (0.1-2.0 micrograms/ml) were continuously extracted by the liver and its extraction ratio at steady-state (Ess) was lowered as the inflow concentration increased. Hepatic clearance of [111In]Gal-BSAs increased significantly according to the increase in the number of galactose residues per BSA at an inflow concentration of 0.7 micrograms/ml. The outflow patterns of [111In]Gal-BSAs at various inflow concentrations were simultaneously fitted to a one-organ pharmacokinetic model, by which we can characterize their binding to the cell surface and internalization processes separately. The parameters obtained were varied significantly among [111In]Gal-BSAs depending on the number of galactose residues and indicate that not only the binding to the receptors but also the internalization after the binding are regulated by the number of galactose residues per BSA during hepatic uptake.     

Expression, function, and regulation of E-type prostaglandin receptors (EP3) in the nonischemic and ischemic pig heart

The action of prostacyclin, prostaglandin E1 (PGE1), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [3H]PGE1, with a Kd of 3.7 nmol/L and a Bmax of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype-selective agonists sulprostone (EP1 and EP3) and M&B 28.767 (EP3) were active, suggesting the presence of an EP3 receptor subtype. PGE1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP3 receptor coupling to an inhibitory G protein (Gi). Additional in vivo experiments showed that intracoronary infusion of PGE1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of beta-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg i.v.), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-gamma-S-induced decrease of [3H]PGE1 binding. These data demonstrate for the first time that myocardial receptors for PGE1 belong to the EP3 subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.     

Relationship between iodine-123-beta-methyl-p-iodophenyl-pentadecanoic acid washout ratio and oxygen consumption in normal and ischemic myocardium

The relationship between oxygen consumption and iodine-123-beta-methyl-p-iodophenyl-pentadecanoic acid (123I-BMIPP) washout at rest and after exercise was investigated in normal and ischemic myocardium. Sixteen healthy volunteers and 14 patients with ischemic heart disease were examined. After injection of 111MBq of 123I-BMIPP, serial single photon emission computed tomography imaging was performed to evaluate washout ratio after 30 min and 1 hour of rest and after exercise. In the volunteers, the mean washout ratio was 3.3 +/- 3.5% after 1 hour of rest and increased during exercise. The exercise washout ratio showed a better correlation with net pressure rate product (net PRP: cumulative values of PRP during exercise) than with the peak PRP. The exercise washout ratio showed a strong correlation with the net PRP in the range from 180 to 300 x 10(3) mmHg. beat/min and a plateau of 10-15%. In the nine ischemic patients with net PRP > or = 300 x 10(3) mmHg.beat/min, the exercise washout ratio values were significantly elevated in normal segments relative to ischemic segments (10.1 +/- 1.9% vs 4.7 +/- 2.9%, p < 0.001). In the five ischemic patients with net PRP < 300 x 10(3) mmHg.beat/min, washout ratio at rest and after exercise did not differ significantly between normal and ischemic segments. 123I-BMIPP washout ratio increased with increased oxygen consumption during exercise in normal myocardium but not in ischemic myocardium. The patient must exercise before fatty acid metabolism can be compared between normal and ischemic myocardium.     

The platelet-activating factor antagonist BN 52021 attenuates hypoxic-ischemic brain injury in the immature rat

Platelet-activating factor (PAF) is overproduced in ischemic brain. Although postischemic PAF antagonist administration protects the mature brain in some models, little is known about the effects of PAF antagonists in the immature brain. We hypothesized that the PAF antagonist BN 52021 would attenuate perinatal cerebral hypoxic-ischemic injury. To elicit focal hypoxic-ischemic brain injury, 7-d-old (P7) rats (n = 111) underwent right carotid ligation, followed by 2.5-3.25 h of hypoxia (fractional concentration of inspired O2 = 0.08). BN 52021 neuroprotection was evaluated in three groups of experiments: 1) 25 mg/kg/dose, 0 and 2 h posthypoxia; 2), 25 mg/kg/dose immediately before and 1 h after hypoxia; and 3) posthypoxia-ischemia treatment with BN 52021 12.5, 25, or 50 mg/kg/dose in 2 doses 0 and 2 h after hypoxia. All experiments included concurrent vehicle-injected controls. To quantitate severity of injury, bilateral regional cross-sectional areas (groups 1 and 2) or hemisphere weights (group 3) were evaluated on P12. Both pre- and posthypoxic treatment with BN 52021 (25 mg/kg/dose, two serial doses) decreased the incidence of cerebral infarction from 90% to about 30% (p < 0.02, Fisher's exact test). Measurement of cross-sectional areas confirmed neuroprotection and indicated some benefit of pre- over posthypoxic-ischemic treatment in hippocampus and cortex. Over the dose range tested, the neuroprotective effect of BN 52021 administration was not dose-dependent. In contrast, BN 52021 did not attenuate N-methyl-D-aspartate-induced hippocampal excitotoxic injury in P7 rats. Either prophylactic or "rescue" administration of PAF antagonists decreases the incidence and severity of brain injury associated with an episode of perinatal cerebral hypoxia-ischemia.     

The renin-angiotensin system and the effect of propranolol upon the cerebral cortical and hypothalamic circulation in hypoxia

The regulatory mechanisms of the cerebral blood flow have preoccupied the physiology department of Cluj since the end of the 4th decade. These studies continued over the last years. The researches progressed from the studies of regulation by blood pressure changes to the nervous regulation and to the metabolic one. This paper's subject is the renin-angiotensin and adrenalin system influence on the changes of cerebral blood flow during the general hypoxic hypoxia and cephalic ischemia. Experiments were performed in 10 dogs anaesthetised with a mixture of chloralose, urethan and morphine. Hypoxic hypoxia was obtained by breathing a mixture of 11% oxygen in nitrogen, in a closed system and cerebral ischemic hypoxia by partial compression of the carotid arteries, after the ligation of the vertebral and thyroid arteries. The arterial blood pressure and the cerebral and hypothalamic blood flow, measured with the heated thermoelement, were registered. The plasma renin activity was tested radioimmunologically before, at 1.5 min, 5, 10 and 15 min, after the beginning of hypoxia. In ischemic hypoxia the experiment was repeated after venous perfusion with propranolol (0.6 mg/kg/h). The systemic blood pressure increased in both forms of hypoxia. The cortical and hypothalamic blood flow increased with the systemic arterial blood pressure. The hypothalamic blood flow remained stable or diminished a little. Propranolol increased the cerebral blood flow during ischemic hypoxia up to 300%. The i.v. administration of angiotensin (1-5 mg/kg) increased the cortical flow, while the hypothalamic flow remained self-regulated. Plasma renin activity increased more in general hypoxic hypoxia, than in cephalic ischemic hypoxia. After propranolol the increase was higher in this hypoxia. Propranolol produced a major activation of the renin-angiotensin system and of the cortical blood flow in ischemic cephalic hypoxia, the renin-angiotensin system being located in the cerebral structure. As well high doses of angiotensin produced cerebral vasodilatation in small cerebral vessels. This effect was found in our experiments in the cortical blood flow too. Our results indicate a beneficial propranolol effect on cortical circulation in ischemic hypoxia.     

Routine carotid endarterectomy without a shunt, even in the presence of a contralateral occlusion

A 10-year prospective experience with routine non-shunting, even in the presence of a contralateral internal carotid artery occlusion, is reviewed. METHOD AND RESULTS: Carotid endarterectomy was performed without a shunt in 654 consecutive patients: group 1, 513 patients with contralateral stenosis of less than 79%: group 11, 74 patients with a greater than 80% contralateral stenosis; and group 111, 67 patients with a contralateral occlusion. Average cross-clamp time was 23 min. Neurological complications occurred within 30 days in 20 (3.0%) patients (10 strokes, seven transient ischemic attacks in group I, one transient ischemic attack in group II, and one stroke and one transient ischemic attack in group III). Immediate postoperative strokes, i.e. those five cases that could be implicated as caused by lack of a shunt, were rare (0.76%). There were five perioperative deaths (0.76%). CONCLUSION: Carotid endarterectomy may be performed safely without a shunt even in the presence of a contralateral occlusion. Age, sex, preoperative indication, anesthetic agent and contralateral stenosis were not associated with an increased risk of postoperative neurological deficit.     

Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.     

Indium-111-DTPA-folate as a potential folate-receptor-targeted radiopharmaceutical

Indium-111-labeled diethylenetriamine pentaacetic acid (DTPA)-folate was evaluated as a radiopharmaceutical for targeting tumor-associated folate receptors. METHODS: Athymic mice were subcutaneously inoculated with approximately 1.8 x 10(6) folate receptor-positive KB (human nasopharyngeal carcinoma) cells, yielding 0.2- to 0.6-g tumors in 15 days, at which time (111)In-DTPA-folate, (111)In-DTPA or (111)In-citrate was administered by intravenous injection. RESULTS: The (111)In-DTPA-folate conjugate afforded marked tumor-specific (111)In deposition in vivo using this mouse model. The involvement of the folate receptor in mediating tumor uptake of (111)In-DTPA-folate was demonstrated by the blocking of tumor uptake by coadministration of free folic acid (intravenous). The (111)In-DTPA-folate also shows folate receptor-mediated uptake and retention in the kidneys, presumably reflecting radiotracer binding to folate receptors of the proximal tubules. In control experiments, the (111)In-citrate radiopharmaceutical precursor was also shown to afford significant tumor uptake of (111)In, but with much poorer tumor-to-background tissue contrast than that obtained with (111)In-DTPA-folate. Unconjugated (111)In-DTPA showed no tumor affinity. CONCLUSION: Indium-111-DTPA-folate appears suitable as a radiopharmaceutical for targeting tumor-associated folate receptors.     

Comparative studies of the in vivo kinetics of simultaneously injected 111In- and 51Cr-labelled human platelets

The kinetics of simultaneously injected 111In- and 51Cr-labelled platelets have been assessed in 40 subjects, 13 of them thrombocytopenic. 4 platelet survival models were applied. The mean life-time (MLT) of 51Cr-platelets from non-thrombocytopenic individuals was found to be slightly, but significantly, longer than that of 111In-platelets by applying linear and exponential models for data fitting. The in vivo recovery (IVR) of 111In-platelets was significantly higher than that of 51Cr-platelets in this patient group when using all 4 models. In the group of thrombocytopenic patients no statistically significant differences in MLT or IVR were found between 111In- and 51Cr-platelets. However, for each of the 11 51Cr-labelled platelet suspensions with the shortest MLT, a longer MLT was observed in the corresponding 111In-platelets, a finding probably related to antibody-induced elution of 51Cr-activity. The same mechanism might be responsible for an increasing 111In-/51Cr-recovery ratio in the early post-injection period. The efficiency of platelet isolation from blood prior to labelling seemed to influence the IVR, inasmuch as the difference in IVR between 111In- and 51Cr-platelets was eliminated in the group where the yield of 111In-platelets surpassed that of the 51Cr-platelets by more than 15%.     

Myocardial injury associated with potassium arrest

The relative efficacy of potassium-induced ischemic arrest using buffered, isosmotic potassium (25 mEq/liter) was compared with hypothermic arrest in an experimental protocol employing an intact canine heart preparation. Myocardial function (LVSW, dp/dt max), serum creatine phosphokinase levels, myocardial perfusion, and light and electron microscopical examination of the heart were assessed in five groups of 5 dogs each. There was one control group (90 minutes of bypass, no anoxia) and four experimental groups, each subjected to 1 hour of ischemic arrest and 30 minutes of reperfusion, comparing normothermic ischemic arrest (NIA), hypothermic ischemic arrest (myocardial temperature less than 25 degrees C) (HIA), normothermic potassium arrest (NKA), and hypothermic potassium arrest (HKA). Myocardial function decreased significantly following NIA and NKA but remained essentially equal in the control, HIA and HKA groups. Serum creatine phosphokinase analysis documented a significant increase in each group of animals: 2,250 mU after NIA, 1,778 mU after NKA, 1,388 mU after HIA, 1,220 mU after HKA, and 838 mU after control bypass. Left ventricular myocardial perfusion was unmeasurably low after NIA, reduced to 111 m/100 gm of tissue/min after NKA, and increased to 165 to 188 ml/100 gm/min in the control, HIA and HKA groups. Electron microscopical studies showed a range of myocardial changes, from probably irreversible damage after NIA to similar but less diffuse changes after NKA, and to potentially reversible changes after HKA and HIA with the least alteration from control after HIA. The results indicate that potassium arrest alone is not as effective as hypothermia in preventing ischemic injury, and the combination of hypothermia with a single 150 cc administration of potassium (25 mEq/liter) does not appear to provide significant additional protection.     

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.     

Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved

A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effect of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose. 31P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na+-H+ exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemia pH in these hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.     

The effect of cytochrome c preparations on the autoregulation of the cerebral blood flow in ischemia

The effect of animal cytochrome C (Ca), biotechnological cytochrome (Cb) and its hemtetradecapeptide (HTDP) on cerebral blood flow autoregulation during rapid decrease of systemic arterial pressure (SAP) was studied in acute experiments on rats. Cytochrome C preparations caused no effect on the autoregulatory responses of the cerebral vessels in animals with normal cerebral circulation. Injection of 5 mg/kg Ca and Cb and 0.8 mg/kg HTDP promoted restoration of the phenomenon of cerebral blood flow autoregulation in ischemic brain damage in change of SAP from 120 to 60 mm Hg. Prophylactic injection of 20 mg/kg Ca and Cb and 3.3 mg/kg HTDP prevented cerebral blood flow autoregulation disturbance caused by transitory brain ischemia.     

Interface characterization of epitaxial Ag Films on Si(100) and Si(111) grown by molecular beam epitaxy

Silver films grown on Si(100), Si(111), and thin oxide layers by molecular beam epitaxy (MBE) have been studied using multiple analysis techniques, including X-ray diffraction (pole-figure method and double-crystal diffractometry), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and MeV He⁺ Rutherford backscattering spectrometry (RBS)/ channeling. Despite the large lattice mismatch (～25 pct) between Ag and Si, high-quality Ag single-crystal films (surface minimum yield χ_min = 6 to 10 pct) were grown on clean Si substrates. Ag(111) texture films were also grown on the oxide layers of Si(100) and Si(111) surfaces. The epitaxial Ag/Si interface was thermally stable up to at least 500 °C. The adhesion between Ag and the oxide layer was poor. The primary defects contained in the Ag/Si(111) were twins. The quantity of twinning depends on the film thickness and the substrate orientation. For both Ag/Si(100) and Ag/Si(111) epitaxial systems, a high-density defect region was contained in the Ag film within ～1000 Å of the interface. Silver films grown on misoriented Si(111) substrates (～4 deg off normal) were misoriented by 0. 5 deg toward the surface normal.     

Mapping the in vivo distribution of herpes simplex virions

We describe a method for labeling enveloped viral particles with a radiotracer, indium-111, allowing labeled viruses to be traced in vivo by nuclear imaging. After initial optimization experiments, a labeling efficiency of 83% (incorporation yield) was achieved for herpes simplex virus (HSV), resulting in a specific activity of 30 microCi/10(9) PFU. The labeling procedure did not significantly reduce the infectivity of the labeled virus and the virus did not release any significant amounts of the radionuclide within 12 hr after labeling. Sequential imaging of animals after intravenous administration of the labeled virus showed fast accumulation in the liver and redistribution from the blood pool (immediately after injection) to liver and spleen (12-24 hr after injection). At 12 hr after injection 7% of the virus-associated (111)In had been eliminated from the body and the remaining organ distribution of the virus was as follows: spleen 2.87 +/- 0.54% ID/g; liver, 2.60 +/- 0.51% ID/g; kidney, 0.98 +/- 0.31% ID/g; lung, 0.57 +/- 0.10% ID/g; [corrected] and lower amounts in other organs. Our results indicate that the described method allows qualitative and quantitative assessment of viral biodistribution in vivo by nuclear imaging.     

Identification of the major sites of phosphorylation in IGF binding protein-3

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier of insulin-like growth factor I and II in the circulation. IGFBP-3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues and neighboring amino acids potentially involved in defining a protein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO-cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by > 80% in the full-length protein and completely abolished phosphorylation in a 17 kDa IGFBP-3 fragment, derived from digestion with EndoProteinase Lys-C. The 17 kDa fragment contained serines 111 and 113. S111A/S113A, a double serine-to-alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of amino acid substitutions rather than lack of phosphorylation. Mutant S111A/S113A, despite being non-phosphorylated and non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP-3.     

Time window for the contribution of the delta-opioid receptor to cardioprotection by ischemic preconditioning in the rat heart

The present study aimed to examine (1) whether the role of the opioid receptor in ischemic preconditioning (PC) is consistent regardless of the duration of ischemic insult and (2) which opioid receptor subtype contributes to PC. In the first series of experiments, the effects of PC, a nonselective opioid receptor antagonist (naloxone), and their combination on the infarct size after various durations of ischemia were assessed. In anesthetized, open-chest rats, the coronary artery was occluded for 20, 30, or 40 minutes to induce infarction and was reperfused for 3 hours, PC was performed with two cycles of 5-minute ischemia followed by 5-minute reperfusion before the sustained ischemia. At 25 minutes before the ischemia, naloxone was injected alone or in combination with subsequent PC. Infarct size was determined by tetrazolium staining and was expressed as a percentage of the risk area size (%IS/RA). In the second series of experiments, the effects of a delta-receptor-selective antagonist, naltrindole (NTI), and a kappa-receptor selective antagonist, nor-binaltrophimine (nor-BNI), on PC before 30-minute coronary occlusion were assessed. In untreated controls, %IS/RA was 53.1 +/- 3.2 after 20 minutes, 67.9 +/- 3.9 after 30 minutes, and 87.8 +/- 2.0 after 40 minutes of ischemia, respectively. PC significantly reduced %IS/RA after 20, 30, and 40 minutes of ischemia to 3.1 +/- 0.8, 12.8 +/- 1.1, and 42.1 +/- 4.3, respectively (P < 0.05 vs. each control). Naloxone (6 mg/kg) partially attenuated the protection afforded by PC when the sustained ischemia was 30 minutes (%IS/RA = 27.4 +/- 4.5; P < 0.05 vs. PC), but this inhibitory effect of naloxone was not detected when the duration of the ischemia was 20 or 40 minutes. NTI (10 mg/kg) also attenuated infarct size limitation by PC after 30 minutes of ischemia (%IS/RA = 25.6 +/- 3.7), but nor-BNI (10 mg/kg) failed to modify infarct size limitation by PC (%IS/RA = 13.3 +/- 3.2). The present results suggest that activation of the opioid delta-receptor partly contributes to preconditioning against infarction in the rat and that there may be a time window (at around 30 minutes after the onset of ischemia) for this opioid receptor-mediated protective mechanism.     

GABA and glutamate levels in the substantia nigra reticulata following repetitive cerebral ischemia in gerbils

Repetitive cerebral ischemia produces more severe damage than a similar single duration insult. We have previously shown that, in gerbils, damage in the substantia nigra reticulata (SNr) is seen with repetitive insults rather than a single insult. We have also shown that there is a progressive decrease in the extracellular GABA in the striatum in the days preceding such damage, speculating that a loss of GABA may be in part responsible for this damage. This study evaluates the GABA levels in the SNr in animals exposed to repetitive ischemic insults. Each animal received a total of three ischemic insults of 3-min duration at hourly intervals. In vivo microdialysis was carried out to analyze the GABA and glutamate dialysate levels on Days 1, 3, 5, 7, and 14 following the ischemic insult. In the control and treated (ischemic) animals, there was a significant increase in the GABA levels with the introduction of nipecotic acid on Days 1, 3, 5, and 14. However, on Day 7 there was a significant attenuation in the GABA response to nipecotic acid in the treated animals in comparison to the controls. The glutamate levels in the treated animals were similar to the control animals on Days 1, 3, 5, and 7. However, on Day 14 the glutamate levels were significantly lower than on previous days. Our experiments for the first time measure extracellular glutamate and GABA responses in the SNr in animals exposed to repetitive ischemic insults. Our experiments show that there is a significant decrease in the GABA concentrations at a time when ischemic damage is developing in this region. This confirms our hypothesis that a decrease in GABA may be one factor contributing to neuronal damage during the period following repetitive ischemic insults. Further, the rebound increase in GABA levels on Day 14 with a concomitant fall in glutamate levels would indicate that reparative processes are still active in the 2 weeks following the insult.