Intracisternal administration of glibenclamide or 5-hydroxydecanoate does not reverse the neuroprotective effect of ketogenic diet against ischemic brain injury-induced neurodegeneration.

PRIMARY OBJECTIVE: To investigate the role of ATP-sensitive potassium (K(ATP)) channels in the neuroprotective effects of a ketogenic diet against cardiac arrest-induced cerebral ischemic brain injury-induced neurodegeneration. RESEARCH DESIGN: Male Sprague Dawley rats were randomly divided into three groups and were fed with a ketogenic diet for 25 days before being subjected to a cardiac arrest-induced cerebral ischemia for 8 minutes 30 seconds. Four hours before cardiac arrest-induced cerebral ischemia, one group was intracisternally injected with glibenclamide, a plasma membrane K(ATP) channel blocker. The second group was injected with 5-hydroxydecanoate, a mitochondrial K(ATP) channel blocker. The third group was without the pre-treatment with K(ATP) channel antagonist. Nine days after the cardiac arrest, rats were sacrificed. Fluoro-jade (FJ) staining was used to evaluate cerebral ischemic neurodegeneration in the rat brain sections. MAIN OUTCOMES AND RESULTS: The number of FJ-positive degenerating neurons in the CA1 area of the hippocampus, the cerebellum and the thalamic reticular nucleus of the ketogenic diet-fed rats with or without glibenclamide or 5-hydroxydecanoate pre-treatment before cardiac arrest-induced cerebral ischemia is zero. CONCLUSIONS: The results suggest that K(ATP) channels do not play a significant role in the neuroprotective effects of the ketogenic diet against cardiac arrest-induced cerebral ischemic injury-induced neurodegeneration.

Ketogenic diet prevents cardiac arrest-induced cerebral ischemic neurodegeneration.

Ketogenic diet (KD) is an effective treatment for intractable epilepsies. We recently found that KD can prevent seizure and myoclonic jerk in a rat model of post-hypoxic myoclonus. In the present study, we tested the hypothesis that KD can prevent the cerebral ischemic neurodegeneration in this animal model. Rats fed a standard diet or KD for 25 days were being subjected to mechanically induced cardiac arrest brain ischemia for 8 min 30 s. Nine days after cardiac arrest, frozen rat brains were sectioned for evaluation of ischemia-induced neurodegeneration using fluoro-jade (FJ) staining. The FJ positive degenerating neurons were counted manually. Cardiac arrest-induced cerebral ischemia in rats fed the standard diet exhibited extensive neurodegeneration in the CA1 region of the hippocampus, the number of FJ positive neurons was 822+/-80 (n=4). They also showed signs of neurodegeneration in the Purkinje cells of the cerebellum and in the thalamic reticular nucleus, the number of FJ positive neurons in the cerebellum was 55+/-27 (n=4), the number of FJ positive neurons in the thalamic reticular nucleus was 22+/-5 (n=4). In contrast, rats fed KD showed no evidence of neurodegeneration, the number of FJ positive neurons in these areas were zero. The results demonstrate that KD can prevent cardiac arrest-induced cerebral ischemic neurodegeneration in selected brain regions.

Brivaracetam is superior to levetiracetam in a rat model of post-hypoxic myoclonus.

In the present study, we evaluated the anti-seizure and anti-myoclonic activity of levetiracetam and brivaracetam in an established rat model of cardiac arrest-induced post-hypoxic myoclonus. We found that brivaracetam (0.3 mg/kg, the minimal effective dose) was more potent than levetiracetam (3 mg/kg, the minimal effective dose) against post-hypoxic seizures. The anti-seizure activity of both compounds occurred 30 min following intraperitoneal (i.p.) administration and was maintained over the entire 150 min post-dose observation period. Both brivaracetam and levetiracetam significantly reduced auditory stimulated post-hypoxic myoclonus from a dose 0.3 mg/kg. At that dose, the anti-myoclonic activity of brivaracetam was already maximal whereas it continued to increase in a dose-relation manner with levetiracetam, suggesting that brivaracetam is a more potent agent. The onset and the duration of anti-myoclonic activity of both compounds were similar. These findings demonstrate that brivaracetam possesses more potent anti-seizure and anti-myoclonic activity than levetiracetam in an established rat model of cardiac arrest-induced post-hypoxic myoclonus.

Effects of heavy metals on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus (Powys).

The effects of Cu, Cd, Zn and Cr on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus were compared. The 96-h LC50 for Cu, Cd, Zn and Cr were 0.36, 1.52, 1.76 and 36.9 mg l(-1), respectively. Four sublethal concentrations of each metal plus a control were prepared and the snails were exposed to experimental solutions for 96 h. Feeding behaviour was studied after the snails were starved for five days. As compared with the control, the number of individuals feeding was significantly reduced by exposure to 0.05 mg l(-1) Cu, 0.2 mg l(-1) Zn, 0.5 mg l(-1) Cd and 5 mg l(-1) Cr. The time spent feeding was greater for individuals exposed to greater concentrations of Zn and Cd but no effect was found for Cu and Cr. Chemoreception of food was studied by placing the snails at a fixed distance of 15 cm from the bait. The success rate of reaching the bait was less for individuals exposed to Cr but no effect was found for Zn, Cu or Cd. The time required for an individual to reach the bait decreased as the concentration of Zn increased. In contrast, a longer time was required for individuals exposed to Cr whereas the effect of Cd and Cu was insignificant. The potential of using feeding behaviour and chemoreception in contaminant evaluations is discussed.

Responses of the green-lipped mussel Perna viridis (L.) to suspended solids.

Laboratory experiments were conducted to investigate the lethal and sublethal effects of suspended solids on the survival and physiological, behavourial and morphological changes of the green-lipped mussel Perna viridis collected from Tolo Harbour, Hong Kong. Results showed that P. viridis survived in all test conditions of suspended solids from 0 to 1,200 mg/l over a period of 96 h. Physiological responses of the green-lipped mussel under 14-d exposure of suspended solids from 0 to 600 mg/l, followed by 14-d recovery in natural seawater, revealed no significant changes (p > 0.05) in oxygen consumption and dry gonosomatic index for treatments in different concentrations of suspended solids and exposure time. Changes in clearance rate were only found to be significant (p < 0.001) with exposure time. Responses in behavourial and morphological changes of the green-lipped mussel were also studied under similar experimental treatments and exposure time. Byssus production was significantly (p < 0.001) related to exposure time. Gill damage, however, was significantly greater in treatments (p < 0.001). Present findings suggested that P. viridis could tolerate a high level of suspended solids in the laboratory. There were dose-dependent effects of suspended solids on morphology of gill filaments. Implications of survival and responses of the green-lipped mussel to suspended solids in the marine environment are discussed.

An N-methyl-D-aspartate receptor channel blocker with neuroprotective activity.

Excitotoxicity, resulting from sustained activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype, is considered to play a causative role in the etiology of ischemic stroke and several neurodegenerative diseases. The NMDA receptor is therefore a target for the development of neuroprotective agents. Here, we identify an N-benzylated triamine (denoted as NBTA) as a highly selective and potent NMDA-receptor channel blocker selected by screening a reduced dipeptidomimetic synthetic combinatorial library. NBTA blocks recombinant NMDA receptors expressed in Xenopus laevis oocytes with a mean IC(50) of 80 nM; in contrast, it does not block GluR1, a glutamate receptor of the non-NMDA subtype. The blocking activity of NBTA on NMDA receptors exhibits the characteristics of an open-channel blocker: (i) no competition with agonists, (ii) voltage dependence, and (iii) use dependence. Significantly, NBTA protects rodent hippocampal neurons from NMDA receptor, but not kainate receptor-mediated excitotoxic cell death, in agreement with its selective action on the corresponding recombinant receptors. Mutagenesis data indicate that the N site, a key asparagine on the M2 transmembrane segment of the NR1 subunit, is the main determinant of the blocker action. The results highlight the potential of this compound as a neuroprotectant.

MinK endows the I(Ks) potassium channel pore with sensitivity to internal tetraethylammonium.

I(Ks) channels are heteromeric complexes of pore-forming KvLQT1 subunits and pore-associated MinK subunits. Channels formed only of KvLQT1 subunits vary from I(Ks) channels in their gating kinetics, single-channel conductance, and ion selectivity. Here we show that I(Ks) channels are more sensitive to blockade by internal tetraethylammonium ion (TEA) than KvLQT1 channels. Inhibition by internal TEA is shown to proceed by a simple bimolecular interaction in the I(Ks) conduction pathway. Application of a noise-variance strategy suggests that MinK enhances blockade by increasing the dwell time of TEA on its pore site from approximately 70 to 370 micros. Mutation of consecutive residues across the single transmembrane segment of MinK identifies positions that alter TEA blockade of I(Ks) channels. MinK is seen to determine the pharmacology of I(Ks) channels in addition to establishing their biophysical attributes.

The conduction pore of a cardiac potassium channel.

Ion channels form transmembrane water-filled pores that allow ions to cross membranes in a rapid and selective fashion. The amino acid residues that line these pores have been sought to reveal the mechanisms of ion conduction and selectivity. The pore (P) loop is a stretch of residues that influences single-channel-current amplitude, selectivity among ions and open-channel blockade and is conserved in potassium-channel subunits previously recognized to contribute to pore formation. To date, potassium-channel pores have been shown to form by symmetrical alignment of four P loops around a central conduction pathway. Here we show that the selectivity-determining pore region of the voltage-gated potassium channel of human heart through which the I(Ks) current passes includes the transmembrane segment of the non-P-loop protein minK. Two adjacent residues in this segment of minK are exposed in the pore on either side of a short barrier that restricts the movement of sodium, cadmium and zinc ions across the membrane. Thus, potassium-selective pores are not restricted to P loops or a strict P-loop geometry.

MinK potassium channels are heteromultimeric complexes.

MinK is a transmembrane protein of 130 amino acids found in the kidney, heart, and vestibular system of mammals. Its expression in Xenopus laevis oocytes induces a voltage-dependent potassium current similar to that seen in vivo. Indirect evidence has fueled speculation that function requires association of MinK and another protein endogenous to oocytes and native tissues. In this report, we show that direct covalent modification of an oocyte membrane protein alters properties of the MinK ion conduction pore; modified channels exhibit decreased potassium conduction and increased permeability to sodium and cesium. The modifying reagents, two membrane-impermeant, sulfhydryl-specific methanethiosulfonate derivatives, react only from the extracellular solution at rates that are determined by the conformational state of the channel. These findings indicate that MinK is intimately associated with an oocyte protein whose exposure to the external solution changes during channel gating and which acts with MinK to establish ion conduction pore function.

MinK residues line a potassium channel pore.

MinK has neither the P region nor signature sequence that characterizes pore-forming subunits of all known K+ channels. A specific minK region has now been identified that affects external blockade by 2 common probes of K+ channel pores. When mutated to cysteine, residues in this region render minK susceptible to covalent blockade by methanethiosulfonate ethylsulfonate and alter reversible inhibition by tetraethylammonium. The 2 blockers are found to share overlapping binding site determinants and to interact. Since inhibition by external tetraethylammonium is sensitive to voltage and to the internal concentration of permeant ions, we argue that tetraethylammonium blocks by occluding the external end of a water-filled transmembrane pore. These findings support the view that minK is directly involved in forming a K+-selective ion conduction pathway.

Lithium attenuates the effects of dynorphin A(1-13) on inositol 1,4,5-trisphosphate and intracellular Ca2+ in rat ventricular myocytes.

When rat ventricular myocytes were stimulated with dynorphin A(1-13), a transient and rapid increase followed by a sustained and prolonged elevation in the intracellular levels of inositol 1,4,5-trisphosphate ¿Ins(1,4,5)P3¿ was observed. The responses were dose-related and abolished by nor-binaltorphimine. In the presence of lithium and absence of extracellular free inositol, the initial rapid elevation in Ins(1,4,5)P3 remained the same, but the second phase of sustained and prolonged elevation was abolished. Under this condition, the elevation in cytosolic free Ca2+ ([Ca2+]i) was reduced significantly although there was still a detectable elevation over a time period when the Ins(1,4,5)P3 was at the basal level. The responses in Ins(1,4,5)P3 and [Ca2+]i were not affected by lithium when stimulation of ventricular myocytes with dynorphin A(1-13) was performed in the presence of extracellular inositol. The data suggest that in rat ventricular myocytes, the kappa-opioid receptor agonist stimulated mobilization of [Ca2+]i was mediated mainly by Ins(1,4,5)P3.

Further characterization of [3H]U69593 binding sites in the rat heart.

K binding sites in the crude membrane preparation of the rat heart homogenate were further characterized by a displacement binding assay of [3H]-U69593 with specific kappa ligands and a direct binding assay with [3H]-etorphine. Scatchard analysis of specific [3H]-U69593 binding showed that the Kd and Bmax were 6.4 +/- 1.0 nM and 97 +/- 8 fmol/mg protein. respectively. The binding of [3H]-U69593 was effectively displaced by the selective kappa 1 ligands, U-69593 and U-50488H, but only weakly displaced by Met5-enkephalin-Arg6-Phe7, a selective kappa 2 ligand, which showed only 11 +/- 3% inhibition of [3H]-U69593 binding at the concentration of 1 microM. In addition, there was no binding site for [3H]-etorphine, known to bind to mu, delta and kappa 2 binding sites, but not kappa 1 binding sites. The findings suggest that the kappa binding sites in the rat heart most likely belong to the kappa 1 subtype. The binding sites have high and low affinity components as nonlinear regression analysis of the competition curves is best fit by two components with IC50 values of 11 +/- 2 and 62 +/- 7 nM for U-69593, and 9.9 +/- 1.5 and 414 +/- 108 nM for U-50488H. Furthermore, the binding of [3H]-U69593 were inhibited by both monovalent cations (Na+, Li+) and divalent cations (Mg2+, Mn2+ and Ca2+).

Signal transduction in the cardiac k-receptor.

The presence of k-binding sites in the heart suggests a regulatory role of k-receptor in the cardiac functions. Recent studies have provided evidence that activation of cardiac k-receptor elevates intracellular free calcium ([Ca2+]i) by increasing mobilization of calcium from the intracellular store. The mobilization of intracellular calcium results from an increased production of inositol-1,4,5-trisphosphate (IP3). The increase in [Ca2+]i may manifest in cardiac arrhythmias while the depletion of calcium from the intracellular store may reduce the contractility elicited upon depolarization. The responses of IP3/[Ca2+]i are significantly attenuated after development of tolerance to k-opioids due mainly to the impairment of postreceptor events. The attenuated responses of IP3/[Ca2+]i to k-receptor activation may be responsible for the failure of the k-agonists to induce cardiac arrhythmias and to reduce electrically induced calcium transients in the ventricular myocytes.

Effects of chronic U50,488H treatment on binding and mechanical responses of the rat hearts.

The effects of chronic injection of U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +), a selective kappa opioid agonist, on the properties of the binding sites of tritiated U69593 [(5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl)benzeneacetamide], another selective kappa opioid agonist, and mechanical responses to U50,488H of the heart were studied. Rats received injection twice a day with U50,488H for 4 days. Binding studies on the crude membrane homogenates revealed that there was no change in maximum binding, but a significant increase in Kd after the treatment, indicating that the number of kappa binding sites remained unchanged whereas the affinity of the binding sites to kappa-agonist decreased. The study on the mechanical responses to U50,488H in the isolated perfused heart preparation showed that although the agonist at 10(-6) M caused MR2266 reversible reductions in heart rate and force of contraction as well as ventricular ectopic beat in the heart of rats in the control group, its effects were absent in the U50,488H-treated group, indicating the development of tolerance to the mechanical effects of U50,488H on the heart. The results indicate that the development of tolerance to the mechanical effects of a kappa-agonist after chronic treatment with the agonist was not accompanied by down-regulation, but only a slight and significant reduction in affinity of kappa binding sites in the rat heart.

Chronic morphine treatment increases the number, but decreases the affinity of [3H]-U69,593 binding sites in the rat heart.

In the present study, the effects of chronic morphine treatment on the binding properties of tritiated U69,593, a specific kappa-ligand, in the rat heart were determined. Adult rats were given morphine through osmotic pumps at a rate of 80 micrograms/hour supplemented by daily injection of morphine at increasing doses for 9 days. The increase in colonic temperature to morphine, used as an indicator of development of tolerance in the rat, was measured daily. It was shown that, on day 7 following morphine treatment, the rats developed tolerance to morphine as indicated by an attenuated hyperthermic response to morphine. The [3H]-U69,593 specific binding properties were determined by direct receptor binding assay. The binding sites increased gradually and reached a significantly higher level at day 10. Scatchard analysis showed that both Bmax and Kd increased at day 10 following morphine treatment, indicating an increase in number of sites and a reduction in affinity to the kappa-ligand. Acute morphine injection at a dose of 10 mg/kg did not cause any significant alteration of [3H]-U69,593 binding sites. Two days after withdrawal of morphine, the [3H]-U69,593 binding sites returned to the original level. The finding of the present study indicates that there is an up-regulation of number, but a reduction in affinity of kappa-binding sites following chronic morphine treatment.

kappa-Opioid receptor stimulation increases intracellular free calcium in isolated rat ventricular myocytes.

The effect of two specific kappa-agonists, dynorphinA1-13 and U50,488H, on intracellular free calcium [Ca]i in isolated rat ventricular myocytes was studied. A spectrofluorimetric method using fura 2 as calcium indicator was employed. It was found that both agonists increased [Ca]i dose-dependently. The effect was attenuated by Mr 2266, a kappa-antagonist, indicating that the effect is a kappa-receptor mediated event. The effect was abolished by pretreatment with ryanodine, a drug that mobilizes calcium from the sarcoplasmic reticulum. It was, however, not affected by nifedipine, a calcium antagonist or removal of external calcium. The results indicate that the increase in [Ca]i due to kappa-opioid receptor stimulation results primarily from mobilization of calcium from an intracellular pool.

Characterization of [3H]U69593 binding sites in the rat heart by receptor binding assays.

The binding properties of [3H]U69593, a selective k-ligand, in rat heart homogenates were characterized by direct and displacement receptor binding assays. It was found that there are substantial specific [3H]U69593 binding sites in the rat heart. They were saturable, reversible and stereospecific. Both association and dissociation rates were monophasic and the Scatchard plot was linear, indicating a homogeneity of binding sites. The Hill coefficient was close to 1, indicating an absence of cooperativity. The Bmax and Kd were 7.91 fmol/mg and 2.92 nM, respectively. The binding sites were most abundant in the right atrium, followed by right ventricle, left ventricle and left atrium in descending order. The study provides information on the properties and distribution of k-binding sites in the rat heart.

Effects of drugs interacting with opioid receptors during normal perfusion or ischemia and reperfusion in the isolated rat heart--an attempt to identify cardiac opioid receptor subtype(s) involved in arrhythmogenesis.

Cardiac opioid receptors have been shown to be involved in the genesis of arrhythmias during ischemia and reperfusion. The present study was aimed at elucidating the receptor subtype(s) involved in arrhythmogenesis. Two series of experiments were conducted. In the first, effects of prototype opioid agonists, namely, (D-Ala2, NMe4, Gly-ol)-Enkephalin (DAGO), U50,488H and (D-Pen2, Pen5)-Enkephalin (DPDPE) and (D-Ala2, D-Leu2)-Enkephalin (DADLE), representing mu-, kappa- and delta-agonists, respectively, in disturbing the normal cardiac rhythm in the isolated perfused rat heart were investigated. Both DAGO and U50,488H were arrhythmogenic, whereas the effects of the delta-agonists, DPDPE and DADLE at a same dose range (44-396 nmol/heart) as that of DAGO were almost negligible. U50,488H was by far the most potent as it induced ventricular arrhythmias including frequent PVC and VT even at a dose (44 nmol/heart) at which other agonists either produced no or negligible effect. In the second series of experiments, the antiarrhythmogenic effects of mu-antagonist (naloxone) and kappa-antagonist (MR 2266) against arrhythmias arising during ischemia and reperfusion were compared. The effects of MR 2266 were significantly greater than that of naloxone. Results of the present study suggest that the cardiac kappa-receptors are the most likely receptor-subtype involved in arrhythmogenesis during ischemia and reperfusion.

Inhibition of metoprolol metabolism by chloroquine and other antimalarial drugs.

The ability of a series of antimalarial drugs to impair the metabolism of metoprolol in rat and man has been examined. Chloroquine was a potent inhibitor in rat liver microsomes (Ki value for metoprolol alpha-hydroxylation = 0.18 microM and for O-demethylation = 0.36 microM). The other antimalarial drugs also inhibited metoprolol oxidation. Quinine was similar to chloroquine in potency, while quinidine, primaquine and mefloquine were slightly less potent. Chloroquine also inhibited metoprolol oxidation in human liver microsomes, although it was about two orders of magnitude less potent than in the rat and the extent of impairment varied greatly between individual livers. Intraperitoneal administration of chloroquine to anaesthetized rats decreased the clearance of metoprolol (40 mg tartrate salt kg-1 i.p.) to 54, 34, 20 and 26% of the control value at doses of 2.5, 4.0, 25 and 40 mg kg-1, respectively. We conclude that antimalarial treatment might have contributed to a previously reported difference in the metabolic pattern of metoprolol between Caucasians and Nigerians.