Moon Seok Park

Chronic nicotine and smoking treatment increases dopamine transporter mRNA expression in the rat midbrain.

Previous pharmacokinetics and electrophysiological results indicated an important role of nicotine in the modulation of dopamine transporter (DAT). To elucidate the expression changes of DAT on chronic nicotine and smoke administration, the effects of nicotine and passive cigarette smoke on DAT mRNA expression in the ventral tegmental area (VTA) and the substantia nigra (SN) area were examined using in situ hybridization and RNase protection assay. The results showed that chronic nicotine and smoke exposure highly unregulated DAT mRNA in the VTA and SN areas, including the dorsal part of substantia nigra pars compacta. Smoke for 30 min showed the highest increasing effect, whereas nicotine and smoke for 10 min only had slightly increasing effects. However, smoke for 1 h showed an increasing effect to a lesser extent than 30 min. These results revealed a new aspect of nicotines modulation on the DAT, and may have important roles in neuropsychological disorders related to the midbrain abnormalities such as drugs addiction.

Dopamine D1 and D2 receptor mRNA up-regulation in the caudate-putamen and nucleus accumbens of rat brains by smoking.

Nicotine, the toxic substance that is exclusively absorbed from smoking, produces a wide array of behavior and collectively propels drug-seeking behaviors when abused. The neurotransmitter dopamine (DA) is important in the reward and reinforcing properties of many addictive drugs: however, the effect of nicotine by cigarette smoking itself on the expression of DA receptors in the caudate-putamen (CPu), nucleus accumbens (NAc) and olfactory tubercle (OTu) has not been elucidated completely. Hence, the effects of smoking and nicotine on DA receptors need to be defined. In this research, the effect of smoking and nicotine addiction on the DA D1 and D2 receptors in the rat CPu, NAc and OTu were studied. Adult male Spraque-Dawley (S.D., n=50) rats were administered with cigarette smoke (passive inhaled for 10 min and 1 h, 500 ml x 3 times/day, 4 weeks) and nicotine (oral, 3 mg/day). DA D1 and D2 receptor mRNA levels were determined by in situ hybridization and RNase protection assay (RPA). In the smoking groups (10 min and 1 h), DA D1 and D2 receptor mRNA greatly increased in the CPu and NAc, and most of all in the NAc. The nicotine treated group showed increased expression of DA D1 and D2 receptor mRNA too, but statistically less than in the smoking group. In the smoking group, DA D1 and D2 receptor mRNA levels were significantly higher in the CPu and NAc than in the nicotine group (P<.01). These results suggest that smoking and nicotine administration both influence DA receptor mRNAs in the CPu, NAc and OTu, in terms of up-regulation. The up-regulation was much more evident in the smoking group than in the nicotine group. In conclusion, we believe that smoking up-regulate the DA receptor mRNA expression significantly higher in rat CPu and NAc than nicotine but only a little bit higher in OTu.

Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor in Bladder Cancer Epithelia and GnRH Effect on Bladder Cancer Cell Proliferation

Introduction: Gonadotropin-releasing hormone (GnRH) is the pivotal hormone in the hypothalamopituitary gonadal axis. Recently, localization of extrahypothalamic GnRHs and GnRH receptors has been made in several organs but not in the bladder. We studied the extrahypothalamic GnRH and GnRH receptor in bladder cancer epithelia, and the role of GnRH on bladder cancer cell proliferation. Material and Methods: Normal human bladder mucosa, human transitional bladder cancer tissue, and 4 bladder cancer cell lines were used. For culture media, normal or charcoal-stripped serum with exogenous GnRH of different concentrations (0, 10–3, 10–5, and 10–7M) were supplemented. For detection of GnRH and the GnRH receptor and its mRNAs, RNase protection assay, in situ hybridization, and immunocytochemistry were performed. Cellular proliferation was measured by hemocytometer and the cell cycle by flow cytometer. Results: GnRH and GnRH receptor mRNA expression were detected in all cancer cell lines and human bladder cancer tissues. GnRH and GnRH receptors were localized in bladder cancer epithelial cells, but the density was not even. There were no significant differences (p > 0.05) in proliferation, and no significant change (p > 0.05) in cell cycle with GnRH supplements compared to controls. Conclusion: The bladder epithelia produce GnRH and the GnRH receptor in both normal and malignant tissues, but GnRH does not induce proliferation or cell cycle change of bladder cancer cells.

Early fetal expression of GABAB1 and GABAB2 receptor mRNAs on the development of the rat central nervous system

GABA(B) receptors are G-protein-coupled receptors that mediate slow onset and prolonged effects of GABA in the central nervous system (CNS). While they appear to influence developmental events, depending on where they are found at a synapse, little, if anything, is known as to the expression of GABA(B1) and GABA(B2) receptor mRNAs during the early developmental stages. We used in situ hybridization and RNase protection assays (RPA) to investigate the early fetal expression of GABA(B1) and GABA(B2) receptor mRNAs on the development of the rat CNS. Our in situ studies defined a pattern of early and strong GABA(B1) receptor mRNA expression in the spinal cord, medullar and cerebral cortex neuroepithelium of discrete brain regions on gestational day (GD) 11.5. On GD 12.5, GABA(B1) receptor mRNAs were found in the hippocampal formation, cerebral cortex, intermediate and posterior neuroepithelium, and the pontine neuroepithelium of whole brain. RPA results showed GABA(B1) receptor mRNA was intensely expressed on GD 11.5 and GD 12.5, when it was first detected in the ganglia, thalamus, and cerebellum. However, GABA(B2) receptor mRNA was not detected on GD 10.5, 11.5, or 12.5. We suggest that GABA(B1) receptor might have a role in the early fetal brain and spinal cord during pre- and post-synaptogenesis, neuronal maturation, proliferation, and migration, and may be more important than the GABA(B2) receptor in the early development of the rat CNS.

Chronic nicotine and smoking exposure decreases GABAB1 receptor expression in the rat hippocampus

Nicotine and smoking have long been proved to play an important role in cognition and memory in the hippocampus. This effect is closely related to the gamma-aminobutyric acid (GABA)ergic system. Previous research has focused on functional and pharmacological aspects of nicotines modulation activity. In this study, the effects of nicotine and different doses of smoking on GABA(B1) expression in the rat hippocampus have been examined using in situ hybridization and RNase protection assay. GABA(B1) receptor mRNAs were intensely expressed in the CA1, CA2, CA3, and dentate gyrus areas of the hippocampus. Nicotine and smoking doses dependently decreased GABA(B1) receptor expression in the hippocampus. These results revealed new aspects of nicotines modulation on GABA(B) receptor, and on learning and memory.

Chronic nicotine and smoke treatment modulate dopaminergic activities in ventral tegmental area and nucleus accumbens and the γ-aminobutyric acid type B receptor expression of the rat prefrontal cortex

Dopaminergic afferents from the mesencephalic areas, such as ventral tegmental area (VTA), synapse with the γ‐aminobutyric acid (GABA)‐ergic interneurons in the prefrontal cortex (PFC). Pharmacological and electrophysiological data show that the reinforcement, the dependence‐producing properties, as well as the psychopharmacologic effects of nicotine depend to a great extent on activation of nicotinic receptors within the mesolimbocortical dopaminergic projection. To explore further the relationship between the mesencephalic dopaminergic neurons and PFC GABAergic neurons, we investigated the effects of nicotine and passive exposure to cigarette smoke on the regulation of tyrosine hydroxylase (TH) in VTA and substantia nigra (SNC) and dopamine (DA) D1 receptor levels in nucleus accumbens (NAc) and caudate‐putamen (CPu). Also, the simultaneous changes in GABAB receptors mRNAs in the PFC were studied. The results showed that chronic nicotine and smoking treatment differentially changed the levels of TH protein in VTA and SNC and DA D1 receptor levels in Nac and CPu. GABAB1 and GABAB2 receptor mRNA levels also showed different change patterns. Ten and thirty minutes of smoke exposure increased GABAB1 receptor mRNA to a greater extent than that of GABAB2, whereas GABAB2 was greatly enhanced after 1 hr of smoke exposure. The TH levels in VTA were closely related to DA D1 receptor levels in NAc and with GABAB receptor mRNA changes in PFC. These results suggest that the mesolimbic pathway and GABAB receptor mRNA in PFC are modulated by nicotine and cigarette smoke, implying an important role in nicotines psychopharmacological effects.

In vivo and in vitro ethanol exposure in prenatal rat brain: GABAB receptor modulation on dopamine D1 receptor and protein kinase A

We have investigated the effects of prenatal ethanol exposure on GABAB receptors (GABABRs), protein kinase A (PKA), and DA D1 receptor (DAD1R) expressions. GABAB1R and GABAB2R showed different age‐dependent expressions in in vivo fetal rat forebrain from gestational days (GD) 15.5 to 21.5 upon 10% ethanol treatment to mother, with and without baclofen at a dose of 10 mg/kg body weight/day. The protein level changes could not be attributed to changes in the level of transcription since GABABR mRNA presented different expression patterns upon in vivo ethanol treatment. Using in vitro cultivated cortical neurons from GD 17.5 fetuses, we also explored the modulatory effects of ethanol on PKA and DAD1R through GABABRs, under 50 μM baclofen and 100 μM phaclofen administrations, with or without 100 mM of ethanol treatment in the culture media. The results showed that 20 min ethanol treatment without baclofen or phaclofen had increasing effects on both the GABABRs. Further, baclofen and phaclofen administration significantly affected PKA and GABABR levels upon 20 min and 1 h ethanol treatment. In contrast, DAD1R showed increasing effects upon ethanol treatment, which was modulated by GABABRs agonist baclofen and antagonist phaclofen. Therefore the present study suggested that the GABABR activity could modulate ethanols cellular effects, which possibly including PKA and DAD1R activities, and may be an underlying cause of ethanols effects. Synapse 62:534–543, 2008.

Ethanol modulates GABAB receptor expression in cortex and hippocampus of the adult rat brain

Using in situ hybridization, RNase protection assay and Western blot, we studied the effects of ethanol on the expression levels of GABA B receptor mRNA and protein in the cortex and hippocampus from adult rat brain. The results showed that ethanol significantly increased GABA B1 and GABA B2 receptor protein expression in the cortex, whereas only GABA B2 was increased in the hippocampus. GABA B receptor agonist baclofen could partially reverse the effect of ethanol. Further studies of the mRNA levels defined that GABA B1 mRNA levels were significantly increased in the hippocampus, with no significant changes of GABA B2 mRNA levels. Moreover, GABA B1 and GABA B2 receptor mRNA levels were increased on 3-week ethanol treatment. Finally, GABA B agonist baclofen and antagonist phaclofen showed significant decreasing effects on GABA B1 receptor mRNA levels in the cortex, but not in the hippocampus. These results were further confirmed by in situ hybridization. Thus, the present results showed the effects of ethanol on GABA B receptors in the cortex and hippocampus, implying the possible role of GABA B receptor in ethanol effects. The effects of GABA B receptor agonist and antagonist suggested that the possible mechanisms underlying that GABA B receptor modulated the behavioral effect induced by ethanol.

Modulation by the GABAB receptor siRNA of ethanol-mediated PKA-α, CaMKII, and p-CREB intracellular signaling in prenatal rat hippocampal neurons

Fetal alcohol syndrome (FAS) is a developmental neuropathology resulting from in utero exposure to ethanol; many of ethanols effects are likely to be mediated by the neurotransmitter γ-aminobutyric acid (GABA). We studied modulation of the neurotransmitter receptor GABABR and its capacity for intracellular signal transduction under conditions of ethanol treatment (ET) and RNA interference to investigate a potential role for GABA signaling in FAS. ET increased GABAB1R protein levels, but decreased protein kinase A-α (PKA-α), calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of cAMP-response element binding protein (p-CREB), in cultured hippocampal neurons harvested at gestation day 17.5. To elucidate GABAB1R response to ethanol, we observed the effects of a GABABR agonist and antagonist in pharmacotherapy for ethanol abuse. Baclofen increased GABABR, CaMKII and p-CREB levels, whereas phaclofen decreased GABABR, CaMKII and p-CREB levels except PKA-α. Furthermore, when GABAB1R was knocked down by siRNA treatment, CaMKII and p-CREB levels were reduced upon ET. We speculate that stimulation of GABAB1R activity by ET can modulate CaMKII and p-CREB signaling to detrimental effect on fetal brain development.

Ethanol modulates the expression of GABAB receptor mRNAs in the prenatal rat brain in an age and area dependent manner

Prenatal ethanol exposure has various deleterious effects on neuronal development. As GABA(B) receptor is known to play an important role during the development of the CNS, we now focused on its mRNA expression pattern in the rat brain during the late gestational days (GD) from 15.5 to GD 21.5. Ethanols effect was also observed from GD 11.5 to GD 21.5. GABA(B1) receptor mRNA showed a high expression level in GD 15.5 and 19.5, while GABA(B2) receptor mRNA did in GD 15.5 and 21.5. The mRNAs levels depended on age and area during development. Ethanol exposure decreased GABA(B1) receptor from GD 11.5 to GD 19.5 with slight increases in GD 21.5. The decreasing effects were area dependent, with the highest effects in the forebrain including cortex, whereas slight effects were observed in the midbrain and hindbrain. The present results suggest an important role of GABA(B) receptor in the effects of ethanol on prenatal brain developmental processes.