Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells

Vitamin C is transported in the brain by sodium vitamin C co‐transporter 2 (SVCT‐2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT‐2 and the uptake and release of [¹⁴C] ascorbate in chick retinal cells. SVCT‐2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT‐2 was expressed in cultured retinal neurons, but not in glial cells. [¹⁴C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium‐free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate‐stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l ‐β‐threo‐benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [³H] d ‐aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2‐Carboxy‐3‐carboxymethyl‐4‐isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7‐initroquinoxaline‐2,3‐dione (DNQX) or (5R,2S)‐(1)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801). However, DNQX, but not MK‐801 or 2‐amino‐5‐phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non‐NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2‐bis (2‐aminophenoxy) ethane‐N′,N′,N′,N′,‐tetraacetic acid tetrakis (acetoxy‐methyl ester) (BAPTA‐AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT‐2, and the release can be stimulated by NMDA or non‐NMDA glutamate receptors.     

Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis

Stimulation of a D₄-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D₄-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparations prelabeled with [³H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D2-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D₃/D₄ DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D₄-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retinas maintained in “eye-cup” preparations. It is suggested that D₄-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D₄ receptor (D₄R) activation promotes light adaptation and suppresses the light‐sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light‐ and D₄R‐signaling pathways converge on the type 1 Ca²⁺/calmodulin‐stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D₄R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D₄R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D₄R, cyclic AMP levels in the dark‐adapted retina are significantly lower compared to wild‐type retina and are unresponsive to light. These changes in Drd4?/? mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity in retinal membranes compared with wild‐type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild‐type mice treated chronically with a D₄R antagonist, L‐745870. Thus, activation of D₄R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross‐talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.     

Dopamine Stimulates K+ Efflux in the Chick Retina via D1 Receptors Independently of Adenylyl Cyclase Activation

Abstract: Dopamine (DA) stimulated K⁺ efflux (assessed as ⁸⁶Rb⁺ efflux) in retinal suspensions of posthatched chicken. This effect was dose dependent (EC₅₀= 22 μM), was mimicked by the D₁-selective antagonist SKF-38393, and reversed by the D₁-selective antagonist SCH-23390, indicating an involvement of D₁ receptors. Analogues of cyclic AMP (CAMP) did not mimic the DA action. Moreover, DA failed to affect cAMP levels, suggesting that adenylyl cyclase (AC) was not involved. In contrast, forskolin (FSK) stimulated both K⁺ efflux and cAMP accumulation in the retina (EC₅₀ of 10 μM for both effects). The FSK-elicited K⁺ efflux was not mimicked by 1,9-dideoxy-FSK (an analogue of FSK that does not activate AC), suggesting that FSK stimulated K⁺ efflux through the activation of AC. Both DA and FSK inhibited Na⁺,K⁺-ATPase activity in the retina. However, the DA-elicited K* efflux was independent of this inhibition, whereas the FSK effect on K⁺ efflux was largely due to the inhibitory action of the diterpene of the ion pump. A possible role of protein kinase C (PKC) in the DA action was explored. The PKC activator 4β-phorbol 12-myristate 13-acetate (4β-PMA) potently (EC₅₀= 4 nM) stimulated K⁺ efflux. This action was not mimicked by the inactive isomer 4α-PMA. When added together, DA and 4β-PMA behaved in an additive manner, suggesting separate mechanisms of action for these two drugs. Moreover, DA failed to stimulate retinal phosphoinositide hydrolysis, a well-known pathway leading to PKC activation. These data suggest that DA acting through D₁ receptors and independently of AC can modulate its target cell excitability in the chick retina by stimulating K⁺ efflux pathways. The mechanism of the DA action remains to be clarified.     

Differential Regulation of D1 Dopamine Receptor- and of A2a Adenosine Receptor-Stimulated Adenylyl Cyclase by μ-, δ1-, and δ2-Opioid Agonists in Rat Caudate Putamen

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D₁ dopamine or A_2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D₁ dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A_2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ-[Tyr-d -Ala-Gly-(N-Me)Phe-Gly-ol (DAMGO)], δ₁-[Tyr-d -Pen-Gly-Phe-d -Pen (DPDPE)], and δ₂- ([d -Ala²]deltorphin-II [DT-II]) opioid agonists on the D₁ dopamine receptor- and A_2a adenosine receptor-stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT-II inhibit forskolin-stimulated adenylyl cyclase [selectively antagonized by d -Phe-Cys-Tyr-d -Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; μ antagonist), 7-benzylidenenaltrexone (BNTX; δ₁ antagonist), and naltriben (NTB; δ₂ antagonist), respectively], but only μ- and δ₂-opioid agonists inhibit D₁ dopamine-stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT-II and DPDPE inhibit A_2a adenosine-stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A_2a adenosine-stimulated adenylyl cyclase activity. These results suggest that μ-, δ₁-, and δ₂-opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ₁ and δ₂ subtypes. μ-Opioid receptors may be preferentially expressed by striatonigral neurons, δ₁- by striatopallidal neurons, and δ₂- by these two striatal efferent neuron populations.     

The subcommissural organ expresses D<Subscript>2</Subscript>, D<Subscript>3</Subscript>, D<Subscript>4</Subscript>, and D<Subscript>5</Subscript> dopamine receptors

Dopamine receptors have been found in certain populations of non-neuronal cells in the brain, viz., discrete areas of ciliated ependyma and the ependymal cells of the choroid plexus. We have studied the presence of both tyrosine-hydroxylase-immunoreactive nerve fibers and dopamine receptors in the subcommissural organ (SCO), an ependymal brain gland that is located in the roof of the third ventricle and that secretes, into the cerebrospinal fluid, glycoproteins that aggregate to form Reissners fiber (RF). Antibodies against D₂, D₃, D₄, and D₅ dopamine receptors were used in immunoblots of bovine striatum, fresh SCO, and organ-cultured SCO, and in immunocytochemistry of the bovine, rat, and mouse SCO. Only a few tyrosine-hydroxylase fibers appeared to reach the SCO. However, virtually all the secretory ependymal and hypendymal cells of the SCO immunoreacted with antibodies against D₂, D₄, and D₅ receptors, with the last-mentioned rendering the strongest reaction, especially at the ventricular cell pole of the secretory ependymocytes, suggesting that dopamine might reach the SCO via the cerebrospinal fluid. The antibodies against the four subtypes of receptors revealed corresponding bands in immunoblots of striatum and fresh SCO. Although the cultured SCO displayed dopamine receptors, dopamine had no apparent effect on the expression of the SCO-spondin gene/protein or on the release of RF-glycoproteins (SCO-spondin included) by SCO explants, suggesting that dopamine affects the function(s) of the SCO differently from the secretion of RF-glycoproteins.Financial support was provided by grants PI 030756 and Red CIEN, Instituto de Salud Carlos III, Spain (to J.M.P.F.), and 1030265 from Fondecyt, Chile (to E.M.R.)     

P2X<Subscript>7</Subscript>R modulation of visually evoked synaptic responses in the retina

P2X₇Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X₇R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X₇R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg²⁺-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X₇R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X₇Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment.     

Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

Muscarinic Inhibition of Hippocampal and Striatal Adenylyl Cyclase is Mainly Due to the M<Subscript>4</Subscript> Receptor

The five muscarinic acetylcholine receptors (M₁–M₅) are differentially expressed in the brain. M₂ and M₄ are coupled to inhibition of stimulated adenylyl cyclase, while M₁, M₃ and M₅ are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2—affinity order M₁ ≥ M₄ >> others—and MT3—highly selective M₄ antagonist—did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M₂, M₄ would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M₄ receptor would be responsible for this regulation. Diana Jerusalinsky and Edgar Kornisiuk contributed equally to this paper.     

Developmental changes in the effects of prostaglandin E<Subscript>2</Subscript> in the chicken ductus arteriosus

Prostaglandin E₂ (PGE₂) is the major vasodilator prostanoid of the mammalian ductus arteriosus (DA). In the present study we analyzed the response of isolated DA rings from 15-, 19- and 21-day-old chicken embryos to PGE₂ and other vascular smooth muscle relaxing agents acting through the cyclic AMP signaling pathway. PGE₂ exhibited a relaxant response in the 15-day DA, but not in the 19- and 21-day DA. Moreover, high concentrations of PGE₂ (≥3 μM in 15-day and ≥1 μM in 19-day and 21-day DA) induced contraction of the chicken DA. The presence of the TP receptor antagonist SQ29,548, unmasked a relaxant effect of PGE₂ in the 19- and 21-day DA and increased the relaxation induced by PGE₂ in the 15-day DA. The presence of the EP receptor antagonist AH6809 abolished PGE₂-mediated relaxation. The relaxant responses induced by PGE₂ and the β-adrenoceptor agonist isoproterenol, but not those elicited by the adenylate cyclase activator forskolin or the phosphodiesterase 3 inhibitor milrinone, decreased with maturation. High oxygen concentrations (95%) decreased the relaxation to PGE₂. The relaxing potency and efficacy of isoproterenol and milrinone were higher in the pulmonary than in the aortic side of the DA, whereas no regional differences were found in the response to PGE₂. We conclude that, in contrast to the mammalian situation, PGE₂ is a weak relaxant agent of the chicken DA and, with advancing incubation, it even stimulates TP vasoconstrictive receptors.     

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian <Emphasis Type="Italic">Dileptus anser</Emphasis>

In some unicellular eukaryotes, cAMP performs functions not only of the secondary messenger, but also of hormone, the primary messenger. We have found that cAMP is bound to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and the enzyme, adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K _D = 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N⁶,O^2′-dibutyryl-cAMP, a non-hydrolyzed cAMP analogue, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated by the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.     

Taurine in adipocytes prevents insulin-mediated H<Subscript>2</Subscript>o<Subscript>2</Subscript> generation and activates Pka and lipolysis

Among many actions assigned to taurine (Tau), the most abundant amino acid in numerous mammalian tissues, it prevents high-fat diet-induced obesity with increasing resting energy expenditure. To sustain this Tau action, the goal of the present study was to explore the acute effects of Tau on baseline and on adrenaline, insulin and their second messengers to modulate lipolysis in white adipose tissue (WAT) cells from rat epididymis. The Tau effects in this topic were compared with those recorded with Gly, Cys and Met. Tau on its own did not modify baseline lipolysis. Tau raised isoproterenol- and dibutyryl-cAMP (Bt₂cAMP)-activated glycerol release. Gly diminished Bt₂cAMP-activated glycerol release, and Cys and Met had no effect. Cyclic AMP-dependent activation of protein kinase A (PKA) in cell-free extracts decreased slightly by Gly and was unaltered by Cys, Met, and Tau. PKA catalytic activity in cell-free extracts was stimulated by Tau and unchanged by Cys, Gly and Met. Gly and Tau effects on PKA disappeared when these amino acids were withdrawn by gel filtration. Insulin-mediated NADPH-oxidase (NOX) raises H₂O₂ pool, which promotes PKA subunit oxidation, and precludes its cAMP activation; thus, lipolysis is diminished. Tau, but not Cys, Gly and Met, inhibited, by as much as 70%, insulin-mediated H₂O₂ pool increase. These data suggested that Tau raised lipolysis in adipocytes by two mechanisms: stimulating cAMP-dependent PKA catalytic activity and favoring PKA activation by cAMP as a consequence of lowering the H₂O₂ pool.     

Frequency-Dependent Modulation of Dopamine Release by Nicotine and Dopamine D<Subscript>1</Subscript> Receptor Ligands: An In Vitro Fast Cyclic Voltammetry Study in Rat Striatum

Nicotine is a highly addictive drug and exerts this effect partially through the modulation of dopamine release and increasing extracellular dopamine in regions such as the brain reward systems. Nicotine acts in these regions on nicotinic acetylcholine receptors. The effect of nicotine on the frequency dependent modulation of dopamine release is well established and the purpose of this study was to investigate whether dopamine D₁ receptor (D₁R) ligands have an influence on this. Using fast cyclic voltammetry and rat corticostriatal slices, we show that D₁R ligands are able to modulate the effect of nicotine on dopamine release. Nicotine (500 nM) induced a decrease in dopamine efflux at low frequency (single pulse or five pulses at 10 Hz) and an increase at high frequency (100 Hz) electrical field stimulation. The D₁R agonist SKF-38393, whilst having no effect on dopamine release on its own or on the effect of nicotine upon multiple pulse evoked dopamine release, did significantly prevent and reverse the effect of nicotine on single pulse dopamine release. Interestingly similar results were obtained with the D₁R antagonist SCH-23390. In this study we have demonstrated that the modulation of dopamine release by nicotine can be altered by D₁R ligands, but only when evoked by single pulse stimulation, and are likely working via cholinergic interneuron driven dopamine release.     

Effects of IP<Subscript>3</Subscript>R2 Receptor Deletion in the Ischemic Mouse Retina

Glial cells in the diseased nervous system undergo a process known as reactive gliosis. Gliosis of retinal Müller glial cells is characterized by an upregulation of glial fibrillary acidic protein and frequently by a reduction of inward K⁺ current amplitudes. Purinergic signaling is assumed to be involved in gliotic processes. As previously shown, lack of the nucleotide receptor P2Y₁ leads to an altered regulation of K⁺ currents in Müller cells of the ischemic retina. Here, we asked first whether this effect is mediated by the IP₃ receptor subtype 2 (IP₃R2) known as the major downstream signaling target of P2Y₁ in Müller cells. The second question was whether lack of IP₃R2 affects neuronal survival in the control and ischemic retina. Ischemia was induced in wild type and IP₃R2-deficient (IP ₃ R2 ^?/?) mice by transient elevation of the intraocular pressure. Immunostaining and TUNEL labelling were used to quantify neuronal cell loss. The downregulation of inward K⁺ currents in Müller cells from ischemic IP ₃ R2 ^?/? retinae was less strong than in wild type animals. The reduction of the number of cells in the ganglion cell layer and of calretinin- and calbindin-positive cells 7 days after ischemia was similar in wild type and IP ₃ R2 ^?/? mice. However, IP₃R2 deficiency led to an increased number of TUNEL-positive cells in the outer nuclear layer at 1 day and to an enhanced postischemic loss of photoreceptors 7 days after ischemia. This implies that IP₃R2 is involved in some but not all aspects of signaling in Müller cells after an ischemic insult.     

多巴胺受体的cDNA克隆

通过多巴胺受体的5个cDNA克隆,综述和分析了5个多巴胺受体(D₁R-D₅R)的基因结构,在染色体上的定位及其mRNA在中枢脑区的分布;比较了这5个受体cDNA克隆的结构特征和药理学性质.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y<Subscript>13</Subscript>-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca²⁺ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y₁₂ and P2Y₁₃ subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC₅₀ values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y₁ receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y₁₂ receptor, suggesting that were mediated by P2Y₁₃-like receptors.¹These authors equally contributed to this work.     

Whole cell bioconversion of vitamin D<Subscript>3</Subscript> to calcitriol using <Emphasis Type="Italic">Pseudonocardia</Emphasis> sp. KCTC 1029BP

Calcitriol is an important drug used for treating osteoporosis, which can be produced from vitamin D₃. The current method of producing calcitriol from vitamin D₃ during cultivation of microbial cells results in low yields of calcitriol and high purification costs. Therefore, in this study, the steps of cell cultivation and bioconversion of vitamin D₃ to calcitriol were separated. Cells of Pseudonocardia sp. KCTC 1029BP were utilized as a whole cell catalyst to produce a high level and yield of calcitriol from vitamin D₃. In addition, the effects of bioconversion buffers, cyclodextrins, and metal salts on the production of calcitriol were comparatively examined and selected for incorporation in the bioconversion medium, and their compositions were statistically optimized. The optimal bioconversion medium was determined as consisting of 15 mM Trizma base, 25 mM sodium succinate, 2 mM MgSO₄, 0.08 % β-cyclodextrin, 0.1 % NaCl, 0.2 % K₂HPO₄, and 0.03 % MnCl₂. Using this optimal bioconversion medium, 61.87 mg/L of calcitriol, corresponding to a 30.94 % mass yield from vitamin D₃, was produced in a 75-L fermentor after 9 days. This calcitriol yield was 3.6 times higher than that obtained using a bioconversion medium lacking β-cyclodextrin, NaCl, K₂HPO₄, and MnCl₂. In conclusion, utilizing whole cells of Pseudonocardia sp. KCTC 1029BP together with the optimal bioconversion medium markedly enhanced the production of calcitriol from vitamin D₃.