Role of N-methyl-D-aspartate receptor in hyperoxia-induced lung injury

Glutamate (Glu) N-methyl-D-aspartate (NMDA) receptor is present in the lungs, and NMDA receptor antagonist MK-801 attenuates oxidant lung injury. We hypothesized that Glu excitotoxicity may participate in the pathogenesis of hyperoxia-induced lung injury. To determine possible pulmonary protective effects, we administered 0.05 ml/kg MK-801 or saline intraperitoneally daily to neonatal rats exposed to more than 95% oxygen in air. After 7 days, MK-801 decreased the hyperoxia-associated elevation of wet-to-dry lung weight, total leukocyte and neutrophil counts, total protein and lactate dehydroase in BAL fluid, total myeloperoxidase activity, and lung pathological injury. MK-801 inhibited hyperoxia-associated increments in reactive oxygen species production and NF-kappaB production. Hence, NMDA receptor antagonist MK-801 ameliorates hyperoxia-induced lung injury in neonatal rats, and is associated with decreased reactive oxygen species and NF-kappaB. We conclude that Glu may play an important role in hyperoxia-induced lung injury by activation of NMDA receptor.     

Characterizing the PK/PD relationship for inhibition of capsaicin-induced dermal vasodilatation by MK-3207, an oral calcitonin gene related peptide receptor antagonist

Aims

Calcitonin gene related peptide (CGRP) receptor antagonists are effective acute migraine treatments. A capsaicin-induced dermal vasodilatation (CIDV) model has been developed to provide target-engagement information in healthy volunteers. In the model, CGRP release is provoked after dermal capsaicin application, by activating transient receptor potential vanilloid-type-1 (TRPV1) receptors at peripheral sensory nerves. Laser Doppler imaging is used to quantify CIDV and subsequent inhibition by CGRP receptor antagonists. We sought to evaluate a CGRP receptor antagonist, MK-3207, in the biomarker model and to assess the predictability of the CIDV response to migraine clinical efficacy.

Methods

An integrated population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the exposure−response relationship for CIDV inhibition by CGRP and TRPV1 receptor antagonists. MK-3207 dose−response predictions were made based on estimated potency from the PK/PD model and mean plasma concentrations observed at the doses investigated.

Results

The results suggested that a 20 mg dose of MK-3207 (EC₅₀ of 1.59 nm) would be required to attain the peripheral CIDV response at a target level that was shown previously to correlate with 2 h clinical efficacy based on phase 3 telcagepant clinical data, and that a plateau of the dose−response would be reached around 40–100 mg. These predictions provided a quantitative rationale for dose selection in a phase 2 clinical trial of MK-3207 and helped with interpretation of the efficacy results from the trial.

Conclusions

The integrated CIDV PK/PD model provides a useful platform for characterization of PK/PD relationships and predictions of dose−response relationships to aid in future development of CGRP and TRPV1 receptor antagonists.     

Brain Neuronal Degeneration Caused by Episodic Alcohol Intoxication in Rats: Effects of Nimodipine, 6,7-Dinitro-quinoxaline-2,3-dione, and MK-801

Rats repeatedly intoxicated with alcohol (ethanol, three times daily) over a 4-day period display neuronal degeneration in the dentate gyrus; entorhinal, piriform, insular, orbital, and perirhinal cortices; and in the olfactory nerve fibers and terminals in the olfactory bulb. Postulating a role for excitotoxicity, we have attempted to prevent the degeneration using antagonists that are neuroprotective in this type of brain damage. In an initial study, continuous subcutaneous infusion of a high dose of the glutamate/NMDA receptor antagonist MK-801 (2 mg/kg/day) by itself caused extensive neuronal degeneration in several brain regions and severe behavioral intoxication that precluded survival if combined with high blood alcohol levels (～300 mg/dl). Moreover, the lower, nonneurotoxic blood alcohol levels (～150 mg/dl) that were compatible with survival worsened the MK-801-induced brain damage. In a subsequent experiment, daily intraperitoneal injections of a lower dose of MK-801 (1 mg/kg/day) resulted in no MK-801 toxicity and, when combined with neurotoxic levels of alcohol, no reduction in alcohol-induced neurotoxicity. Nimodipine, a voltage-gated Ca²⁺ channel blocker, reduced the neuronal damage in the dentate gyrus, but greatly increased it in the piriform cortex when administered intragastrically at 600 mg/kg/day; it provided no protection from alcohol-dependent degeneration when given intragastrically at 100 mg/kg/day. Continuous intracere-broventricular delivery of 0.24 to 0.29 mg/day of 6,7-dinitro-quinoxa-line-2,3-dione, a glutamate/α-amino-3-hydroxy-5-methyl-4-isoxazole receptor antagonist, failed to diminish alcohol-dependent neuronal damage in any brain region. We conclude that brain damage from episodic “binge” alcohol intoxication is not primarily mediated by excitotoxic mechanisms, implying that other, nonexcrtotoxic pathophysiological mechanisms, are involved. Furthermore, MK-801, far from protecting from the alcohol-induced damage, at high doses causes widespread neuropathology that is significantly potentiated by alcohol.     

Alertness and psychomotor performance effects of the histamine-3 inverse agonist MK-0249 in obstructive sleep apnea patients on continuous positive airway pressure therapy with excessive daytime sleepiness: a randomized adaptive crossover study

Objectives

We aimed to evaluate the efficacy of the selective H3 receptor inverse agonist MK-0249 to treat excessive daytime sleepiness (EDS).

Methods

In this three-period, double-blind, crossover study, 125 patients (100 men, 25 women; mean age, 48.6 years) with obstructive sleep apnea receiving nasal continuous positive airway pressure therapy who had refractory EDS were randomized to 2 weeks each of daily MK-0249 (5, 8, 10, or 12 mg, adaptively assigned), modafinil 200 mg, and placebo. At baseline and after each treatment period, six maintenance of wakefulness tests (MWT) and Psychomotor Vigilance Tasks (PVT) were conducted at 2-h intervals, beginning 1 h postdose (∼09:00). The Epworth sleepiness scale (ESS), Clinical Global Impression of Severity (CGIS) and Digit Symbol Substitution Test (DSST) also were assessed. The primary end point was MWT sleep latency averaged over the first four time points (MWT-early).

Results

MWT-early mean change from baseline sleep latency at week 2 was 1.2 min for placebo, 2.1 min for MK-0249 (top two doses pooled; P > .05 vs placebo), and 5.9 min for modafinil (P ? .001 vs placebo). MK-0249 showed improvements vs placebo on secondary and exploratory end points of ESS, CGIS, PVT, and DSST. Insomnia adverse events (AEs) were greater for MK-0249 (combined doses, 17.5%) than for placebo (0.9%) or modafinil (1.8%).

Conclusion

MK-0249 did not significantly affect MWT sleep latency. However, the pattern of improvement on subjective ratings and psychomotor performance end points suggested that MK-0249 was associated with changes in aspects of cognition and performance not captured by the MWT.     

Differentiation renders susceptibility to excitotoxicity in HT22 neurons

HT22 is an immortalized mouse hippocampal neuronal cell line that does not express cholinergic and glutamate receptors like mature hippocampal neurons in vivo.This in part prevents its use as a model for mature hippocampal neurons in memory-related studies.We now report that HT22 cells were appropriately induced to differentiate and possess properties similar to those of mature hippocampal neurons in vivo,such as becoming more glutamate-receptive and excitatory.Results showed that sensitivity of HT22 cells to glutamate-induced toxicity changed dramatically when comparing undifferentiated with differentiated cells,with the half-effective concentration for differentiated cells reducing approximately two orders of magnitude.Moreover,glutamate-induced toxicity in differentiated cells,but not undifferentiated cells,was inhibited by the N-methyl-Daspartate receptor antagonists MK-801 and memantine.Evidently,differentiated HT22 cells expressed N-methyl-D-aspartate receptors,while undifferentiated cells did not.Our experimental findings indicated that differentiation is important for immortalized cell lines to render post-mitotic neuronal properties,and that differentiated HT22 neurons represent a better model of hippocampal neurons than undifferentiated cells.     

Double-tracer autoradiographic study of protein synthesis and glucose consumption in rats with focal cerebral ischemia

Abstract

A double-tracer autoradiographic method for simultaneous measurement of regional glucose utilization (rCMR_glc) and regional protein synthesis (PS) in consecutive brain sections is described and applied to study the metabolism of the ischemic penumbra 2 h after occlusion of the middle cerebral artery (MCAO) in rats. In halothane anesthesia, the left middle cerebral artery was permanently occluded. Two hours after MCAO an i.v. bolus injection of ¹⁴C-deoxyglucose and ³H-leucine was given and circulated for 45 min. Two sets of brain sections were processed for quantitative autoradiography. Neighboring brain sections exposed an X-ray film (3 H-insensitive), and a ³H-sensitive for determination of rCMR_glc and PS, respectively. Sections for PS determination were washed in trichloroacetic acid (TCA) prior to film exposure in order to remove ¹⁴C-deoxyglucose and unincorporated ³H-leucine. Regional rates of PS and glucose utilization were measured by densitometric image analysis. Normal rates of metabolism were defined as mean ± 2 SD of values in the non-ischemic cortex. The volumes of ischemic cortex displaying normal rates of PS and glucose utilization, respectively, were measured. The cortical volume with normal PS was significantly less than that of normal rCMR_glc: 142 (127-147) mm³ vs. 203 (184-206) mm³. Treatment with the glutamate antagonists MK-801 (1 mgkg^–1) and NBQX (30 mg kg^–1 x 2) did not significantly change this, although MK-801 tended to reduce the size of the metabolic penumbra calculated as the difference between ischemic cortex with reduced PS and ischemic cortex with reduced rCMReic. [Neurol Res 1991; 21: 687–694]     

RTP801 immunoreactivity in retinal ganglion cells and its down-regulation in cultured cells protect them from light and cobalt chloride

RTP801, a stress-related protein, is activated by adverse environmental conditions and inhibits the activity of mammalian target of rapamycin (mTOR) in promoting oxidative stress-dependent cell death. RTP801 exists both in the mammalian retina and the lens of the eye. Here, we observed RTP801 immunoreactivity in some retinal ganglion cells. Intravitreal injection of cobalt chloride (CoCl₂) to mimick hypoxia influenced retinal GFAP (glial fibrillary acidic protein) and heme oxygenase-1 (HO-1) levels, but did not affect RTP801 immunoreactivity or mRNA content relative to GAPDH. However, RTP801 mRNA was elevated when compared with Brn3a mRNA, suggesting that RTP801 is activated in stressed Brn3a retinal ganglion cells. In cultures of RGC-5 cells, RTP801 immunoreactivity was located in the cytoplasm and partly present in the mitochondria. An insult of blue light or CoCl₂ increased RTP801 expression, which was accompanied by cell death. However, in cultures where RTP801 mRNA was down-regulated, the negative influence of blue light and CoCl₂ was blunted. Rapamycin nullified the CoCl₂-induced up-regulation of RTP801 and attenuated cell death. Moreover, rapamycin was non-toxic to RGC-5 cells, even at a high concentration (10 μM). The protective effect of rapamycin on RGC-5 cells caused by the inhibition of RTP801 suggests that rapamycin might attenuate retinal ganglion cell death in situ, as in glaucoma.     

Activation of medial prefrontal cortex neurons by phencyclidine is mediated via AMPA/kainate glutamate receptors in anesthetized rats

Phencyclidine (PCP) is a psychotomimetic drug that elicits schizophrenia-like symptoms in healthy individuals, and animals administered PCP are now considered a reliable pharmacological model of schizophrenia. Recent studies have shown that systemically administered PCP produces long-lasting activation of medial prefrontal cortex (mPFC) neurons, and that hyperactivation of mPFC neurons plays a critically important role in the development of PCP-induced behavioral abnormalities. However, the receptors mediating this mPFC activation have not been clearly determined. Here, we examined the effects of local application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA/kainate glutamate receptor antagonist, scopolamine, a muscarinic acetylcholine receptor antagonist, and mecamylamine, a nicotinic acetylcholine receptor antagonist, on the increase in firing rate of mPFC neurons induced by systemic PCP in anesthetized rats. After tonic activation of mPFC neurons by PCP had been established, CNQX, scopolamine, or mecamylamine was iontophoretically applied or pressure-ejected on the recorded neuron. CNQX suppressed PCP-induced elevation of firing rate to baseline level, though scopolamine and mecamylamine each induced little change in firing rate. These findings suggest that PCP-induced activation of mPFC neurons is mediated primarily via AMPA/kainate glutamate receptors.