Mete Ozcan

Inhibitory effects of melatonin on neural lipid peroxidation induced by intracerebroventricularly administered homocysteine

Abstract: Melatonin, the main secretory product of the pineal gland, has been shown to be potentially effective in prevention of numerous types of neurodegenerative disorders in which free radical processes are involved. Homocysteine (Hcy), an independent risk factor for atherosclerosis, undergoes auto‐oxidation and generates reactive oxygen species. The purpose of this study was to test whether intracerebroventricular (ICV) injection of Hcy leads to neural lipid peroxidation and also to investigate the protective effects of melatonin on the brain tissue from oxidative stress of Hcy. Adult male Wistar rats under anaesthesia were injected ICV with Hcy at a dose of 143 μg/kg. Melatonin was administered intraperitoneally to a group of rats for three consecutive days before Hcy injection. The rats were decapitated and brain tissues were removed and hippocampus, cortex and cerebellum were dissected. There was a significant development of oxidative stress as indicated by an increase in malondialdehyde in hippocampus, cortex and cerebellum of rats injected with Hcy, whereas melatonin prevented the elevation of lipid peroxidation. Furthermore, melatonin significantly increased glutathione levels and glutathione peroxidase activity in all brain regions. The present study demonstrates that Hcy, in high levels, may be a causal factor in generation of free radicals in the brain and it may be one of the mechanisms which cause neurodegeneration in elderly people. It also shows that melatonin could potentially be beneficial in prevention of neurodegeneration caused by hyperhomocysteinemia.

Melatonin inhibits high voltage activated calcium currents in cultured rat dorsal root ganglion neurones.

The actions of melatonin on high-voltage activated calcium channels (HVACC) and intracellular free Ca(2+) concentration in cultured dorsal root ganglion (DRG) neurones from neonatal rats were investigated using the whole-cell patch clamp and the fura-2 fluorescence ratio Ca(2+)-imaging techniques. HVACC were pharmacologically and biophysically isolated and the effects of melatonin were investigated. Extracellular application of melatonin inhibited HVACC in a dose dependent manner. In calcium imaging experiments, application of extracellular recording medium containing 30 mM KCl evoked increases in intracellular free Ca(2+) that were dependent upon external Ca(2+) ions. This increase was prevented by both low (10 microM) and high dose (100 microM) of melatonin pre-treatment. The results of this study indicate that the pineal hormone melatonin has inhibitory actions on voltage dependent calcium entry in cultured rat DRG neurones.

Mu opioid modulation of oxytocin secretion in late pregnant and parturient rats: Involvement of noradrenergic neurotransmission

We have investigated effects of µ- and ĸ-opioid agonists and antagonists on plasma oxytocin levels and noradrenaline content in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of 20-day pregnant rats. β-Endorphin, oxytocin, estrogen and progesterone profiles in late pregnant and parturient rats were also sought. Stage of estrous cycle was monitored by vaginal smear, and pro-estrous animals were left overnight with male. In the first set of experiments, pregnant rats were monitored and decapitated on days 20 and 21 and after the delivery of second pup. In the second set, 20-day pregnant rats were intracerebroventricularly infused with morphine (50 µg/10 µl), U50,488H (ĸ-agonist; 50 µg/10 µl), clocinnamox (µ-antagonist; 50 µg/10 µl) and norbinaltorphimine (ĸ-antagonist; 50 µg/10 µl). Controls received saline alone. Serum estrogen and progesterone levels were measured by enzyme immunoassay, and plasma oxytocin and β-endorphin by radioimmunoassay. Noradrenaline and its metabolite (3,4-dihydroxyphenylglycol) were determined in micropunched hypothalamic nuclei by HPLC-ECD. In parturient rats, oxytocin levels were increased (p < 0.05) and β-endorphin decreased (p < 0.01) compared to 20-day pregnant animals. Serum progesterone concentrations progressively declined towards parturition (p < 0.001). Clocinnamox raised oxytocin levels (p < 0.01) while U50,488H caused decreases (p < 0.05). Noradrenaline content was elevated by clocinnamox in the SON (p < 0.01) and PVN (p < 0.05) compared to control values. Other agonists and antagonists had no significant effect on the noradrenergic neurotransmission or oxytocin secretion. We suggest that noradrenaline may mediate the inhibitory effects of µ-opioids on oxytocin release. Our findings have also shown that ĸ-opioid receptors are not involved in modulation of oxytocin neurons in late pregnant rats.

Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones.

Kisspeptins, which are alternatively called as metastin since they were originally identified as products of metastasis suppressor gene KiSS-1, are the natural ligands for the G protein-coupled receptor 54 (GPR54). Kisspeptins are the most potent activators of hypothalamic-pituitary-gonadal (HPG) axis reported to date. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Therefore, the pattern of kisspeptin-induced intracellular signaling and the role of PKC in the intracellular signaling cascade were investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations ([Ca(2+)](i)). The changes in [Ca(2+)](i) were significantly attenuated by pre-treatment with protein kinase C inhibitor. The compatibility of appeared mirrored-patterns of kisspeptin-10-induced changes in [Ca(2+)](i) concentrations in these neurons and GnRH secretion confirm the importance of intracellular calcium flux downstream from GPR54 through PKC signaling pathway.

Leptin modulates noradrenaline release in the paraventricular nucleus and plasma oxytocin levels in female rats: a microdialysis study.

The neural control and mutual interrelationships among individual factors involved in the regulation of food intake and simultaneously related to reproduction are far from being understood. We have suggested that at least some of the effects of orexigenic and anorexigenic peptides might be mediated via noradrenaline release in the paraventricular nucleus (PVN). The main hypothesis was that leptin has an inhibitory action on oxytocin secretion and hypothalamic release of noradrenaline. Non-pregnant female rats in their diestrus were subjected to cannulation of the carotid artery and a microdialysis procedure with the probes in the hypothalamic PVN. Intra-arterial administration of cholecystokinin-8 (CCK) at the dose of 50 mg/kg was used to induce oxytocin and noradrenaline release. Leptin (10 mg/5 ml) was intracerebroventricularly injected in addition to CCK. Blood and microdialysis samples were collected at 20-min intervals for 80 min. Central administration of leptin significantly reduced both plasma oxytocin and hypothalamic noradrenaline responses to CCK at 20 min following the treatments. In conclusion, leptin may inhibit oxytocin secretion by lowering noradrenergic neurotransmission in the PVN. The modulator effect of leptin on noradrenaline release in the PVN may be related to feeding behavior.

Kisspeptin antagonist prevents RF9-induced reproductive changes in female rats

The aim of this study was to determine the modulatory effects of peptide 234 (p234) (an antagonist of GPR54 receptors) on kisspeptin and RF9 (an RFamide-related peptide antagonist)-induced changes in reproductive functions and energy balance in female rats. Female Sprague-Dawley rats were weaned on postnatal day (pnd) 21. The animals were intracerebroventricularly cannulated under general anesthesia on pnd 23. Groups of female rats were injected with kisspeptin, RF9, p234, kisspeptin plus p234, or RF9 plus p234, daily. The experiments were ended on the day of first diestrus following pnd 60. Kisspeptin or RF9 alone advanced vaginal opening (VO), which was delayed by administration of kisspeptin antagonist alone. In the rats given kisspeptin plus p234 or RF9 plus p234, VO was not different from control rats. Kisspeptin and RF9 elicited significant elevations in circulating LH levels. Coadministrations of kisspeptin or RF9 with p234 decreased LH levels significantly. The use of p234 alone did not cause any significant change in LH secretion. Kisspeptin decreased both food intake and body weight while RF9 decreased only food intake without affecting body weight. The effects of kisspeptin on energy balance were also reversed by central administration of p234. In conclusion, kisspeptin antagonist, p234, modulates the effects of kisspeptin on reproductive functions and energy balance, whereas RF9 seems to exert only its effects on reproductive functions by means of GPR54 signaling in female rats.

Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1-7 GnRH neurons.

Melatonin is suggested to have effects on hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Calcium is a second messenger involved in hormone release. Therefore, investigation of the effects of melatonin on intracellular free calcium levels ([Ca(2+)](i)) would provide critical information on hormone release in immortalized GnRH neurons. The pattern of melatonin-induced intracellular calcium signaling was investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Melatonin caused a significant increase in [Ca(2+)](i,) which was greatly blocked by luzindole, a melatonin antagonist, or attenuated by pre-treatment with protein kinase C inhibitor. This study suggests that melatonin seems to have a direct effect on GnRH neurons.

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca2+]i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5 μM Fura-2/AM and [Ca2+]i transients for stimulation with 30 mM KCl (Hi K+) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380 nm, emission was recorded at 510 nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca2+]i transients in a reversible manner. [Ca2+]i increase evoked by Hi K+ was significantly reduced to 99.9 ± 5.1% (n = 18) and 62.5 ± 4.2% (n = 15, P < 0.05) after the application of 5 and 50 µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K+-induced [Ca2+]i responses, reduced to 98.7 ± 4.8% (n = 10) and 69.5 ± 4.5% (n = 9, P < 0.05) inhibition of fluorescence ratio values of Hi K+-induced responses at 5 and 50 μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.

Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons

Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2–based ratiometric Ca2+-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 µM, 100 µM and 300 µM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca2+ imaging protocols revealed that HiK+ (30 mM)-induced intracellular free Ca2+ ([Ca2+]i) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 µM, 100 µM and 300 µM LEV (respectively), without any significant effect on basal levels of [Ca2+]i. This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca2+, providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.

Effects of rosuvastatin on ADMA, rhokinase, NADPH oxidase, caveolin-1, hsp 90 and NFkB levels in a rat model of myocardial ischaemia-reperfusion : cardiovascular topic

Summary Aim Endothelial dysfunction, oxidative stress and inflammation are among the most important mechanisms of ischaemia–reperfusion (I/R) injury. Besides their cholesterol-lowering effects, statins are known to provide protection against myocardial dysfunction and vascular endothelial injury via nitric oxide-dependent mechanisms. The aim of this study was to investigate the effects of rosuvastatin on certain intermediates involved in the generation of nitric oxide (asymmetrical dimethyl arginin, ADMA, caveolin-1 and hsp 90), oxidative stress (rhokinase, NADPH oxidase) and inflammation (NFkB), using an in vivo model of myocardial infarction in the rat. Methods Adult male Sprague Dawley rats were divided into three groups (control, I/R and I/R after 15 days of rosuvastatin administration). Reperfusion was applied for 120 min following left anterior descending coronary artery ischaemia for 30 min. Caveolin-1, hsp 90 and NFkB levels were evaluated with the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and ADMA, rhokinase and NADPH oxidase levels were evaluated with ELISA. Results While NFkB and hsp 90 levels were higher in the I/R group, their levels were significantly lower in the rosuvastatin group. While ADMA and NADPH oxidase levels significantly increased with I/R, they were lower in the rosuvastatin-treated group, but not statistically significant. Rhokinase levels were significantly lower in the rosuvastatin group. Caveolin-1 levels were not different between the groups. Conclusion Our results suggest that ADMA, rhokinase, NADPH oxidase, hsp 90 and NFkB could facilitate I/R injury, and rosuvastatin significantly reduced levels of these parameters. These results indicate that rosuvastatin may have a protective role in I/R injury via mechanisms targeting inflammation, endothelial dysfunction and oxidative stress.