5-HT<Subscript>6/7</Subscript> Receptor Antagonists Facilitate Dopamine Release in the Cochlea via a GABAergic Disinhibitory Mechanism

In humans, serotonin (5-HT) has been implicated in numerous physiological and pathological processes in the peripheral auditory system. Dopamine (DA), another transmitter of the lateral olivocochlear (LOC) efferents making synapses on cochlear nerve dendrites, controls auditory nerve activation and protects the sensory nerve against overactivation. Using in vitro microvolume superfusion techniques we tested 5-HT₆ and 5-HT₇ receptor antagonists whether they can influence dopamine (DA) release from the guinea-pig cochlea in control and in ischemic conditions using currently available and new 5-HT₆ and 5-HT₇ antagonists and mixed antagonists, which were synthesized and characterized for the current study. While the 5-HT₇ antagonist SB-258719 was ineffective, SB-271046, which blocks the 5-HT₆ receptor, caused a significant increase in cochlear DA release what is contradictory with the excitatory nature of this type of receptor. Moreover, the mixed 5-HT_6/7 antagonist EGIS-12233 induced an even more pronounced increase in the resting DA release. To understand why the block of an excitatory receptor results in an increase instead of a decrease in function, we investigated the possible involvement of an indirect neural mechanism through an inhibitory system. In the presence of the GABA_A receptor blocker bicuculline, EGIS-12233 failed to increase the release of DA, suggesting that the serotonin receptor modulation of DA release from the lateral olivocochlear efferents in the cochlea was produced indirectly by decreasing the GABAergic inhibitory tone on dopaminergic nerve endings. The mixed 5-HT₇/D₄ receptor antagonist EGIS-11983 significantly increased both the stimulation-evoked and the resting DA release, while the selective D4 blocker L-741,741 alone had no significant effect. Ischemia, simulated by oxygen and glucose deprivation from the perfusion solution had no action on the effect of the drugs. Drugs that can increase the release of DA from LOC terminals in the cochlea may have a role in the treatment of sensorineural hearing loss.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

Effects of cathepsins B and L inhibition on postischemic protein alterations in the brain

The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN₂, cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.     

Neuroglobin: enzymatic reduction and oxygen affinity

Neuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, involved in neuroprotection. O₂ equilibrium measurements on mouse Ngb yielded significantly different P₅₀ values, ranging from ∼2 torr to ∼10 torr. By a kinetic approach minimizing the effects of protein autoxidation, we measured P₅₀ = 2.2 torr at 20 °C. As predicted from the structure, O₂ binds to the Y44D Ngb mutant more quickly (k = 2.2 s⁻¹ vs 0.15 s⁻¹) and with slightly higher affinity (P₅₀ = 1.3 torr) than wild-type. In addition, we introduced a novel reduction protocol for metNgb based on NADH:flavorubredoxin oxidoreductase (FlRd-red) from Escherichia coli, a candidate for the Ngb reducing activity recently identified in E. coli extracts. Interestingly, E. coli FlRd-red shares sequence similarity with the FAD-binding domain of the human apoptosis-inducing factor, a finding which may have unexpected significance with reference to the mechanism of neuroprotection by Ngb.     

In vitro neuroprotection against glutamate-induced toxicity by pGlu-Glu-Pro-NH2 (EEP)

EEP is a tripeptide structurally similar to thyrotropin releasing hormone (TRH) and, like TRH, it is found in the mammalian brain. TRH has been found to increase in brain regions after seizures and to be neuroprotective. EEP has also been shown to increase in brain regions following seizure activity. We therefore sought to determine whether the similarities between these two peptides might be extended to include neuroprotection. Both TRH and EEP were found to be neuroprotective in vitro against an excitotoxic insult. Interestingly, the two tripeptides appeared to have different mechanisms of action. Even though EEP was as much as four times more neuroprotective than TRH, its ability to reduce glutamate-stimulated increases in intraneuronal Ca²⁺ was about half that of TRH.     

Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l -kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K _m of 2.0 m M and 10.0 m M for l -kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l -glutamine, l -phenylalanine, and l -tryptophan, using either pyruvate (1 m M ) or 2-oxoisocaproate (1 m M ) as a cosubstrate. l -Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K _i = 480 µ M ) and competitively with regard to l -kynurenine ( K _i = 200 µ M ). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.     

Evaluation of nicotine and cotinine analogs as potential neuroprotective agents for Alzheimer’s disease

The currently available therapies for Alzheimer’s disease (AD) and related forms of dementia are limited by modest efficacy, adverse side effects, and the fact that they do not prevent the relentless progression of the illness. The purpose of the studies described here was to investigate the neuroprotective effects of the nicotine metabolite cotinine as well as a small series of cotinine and nicotine analogs (including stereoisomers) and to compare their effects to the four clinically prescribed AD therapies.     

The Neuroprotective Adenosine-Activated Signal Transduction Pathway Involves Activation of Phospholipase C

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid (IAA) followed by “reperfusion” (IAA-R insult), results in severe cytotoxicity, which could be markedly attenuated by prior activation of the adenosine A₁ receptors. We also have demonstrated that adenosine activates a signal transduction pathway (STP), which involves activation of PKCε and opening of K_ATP channels. Here, we provide proof for the involvement also of phospholipase C (PLC) in the neuronal protective adenosine-activated STP. R-PIA, a specific A₁ adenosine receptor agonist, was found to enhance neuronal PLC activity and protect against the IAA-R insult. The PLC inhibitor U73122, abrogated both R-PIA-induced effects. These results demonstrate that activation of PLC is a vital step in the neuronal protective adenosine-induced STP.