Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 were blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.     

Lack or Inhibition of Dopaminergic Stimulation Induces a Development Increase of Striatal Tyrosine Hydroxylase-Positive Interneurons

We examined the role of endogenous dopamine (DA) in regulating the number of intrinsic tyrosine hydroxylase-positive (TH⁺) striatal neurons using mice at postnatal day (PND) 4 to 8, a period that corresponds to the developmental peak in the number of these neurons. We adopted the strategy of depleting endogenous DA by a 2-day treatment with α-methyl-p-tyrosine (αMpT, 150 mg/kg, i.p.). This treatment markedly increased the number of striatal TH⁺ neurons, assessed by stereological counting, and the increase was highly correlated to the extent of DA loss. Interestingly, TH⁺ neurons were found closer to the clusters of DA fibers after DA depletion, indicating that the concentration gradient of extracellular DA critically regulates the distribution of striatal TH⁺ neurons. A single i.p. injection of the D1 receptor antagonist, {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (0.1 mg/kg), the D2/D3 receptor antagonist, raclopride (0.1 mg/kg), or the D4 receptor antagonist, L-745,870 (5 mg/kg) in mice at PND4 also increased the number of TH⁺ neurons after 4 days. Treatment with the D1-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 (10 mg/kg) or with the D2-like receptor agonist, quinpirole (1 mg/kg) did not change the number of TH⁺ neurons. At least the effects of {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 were prevented by a combined treatment with {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393. Immunohistochemical analysis indicated that striatal TH⁺ neurons expressed D2 and D4 receptors, but not D1 receptors. Moreover, treatment with the α4β2 receptor antagonist dihydro-β-erythroidine (DHβE) (3.2 mg/kg) also increased the number of TH⁺ neurons. The evidence that DHβE mimicked the action of {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 in increasing the number of TH⁺ neurons supports the hypothesis that activation of D1 receptors controls the number of striatal TH⁺ neurons by enhancing the release of acetylcholine. These data demonstrate for the first time that endogenous DA negatively regulates the number of striatal TH⁺ neurons by direct and indirect mechanisms mediated by multiple DA receptor subtypes.     

Dopamine Increases CD14+CD16+ Monocyte Migration and Adhesion in the Context of Substance Abuse and HIV Neuropathogenesis

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14⁺CD16⁺ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14⁺CD16⁺ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14⁺CD16⁺ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14⁺CD16⁺ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14⁺CD16⁺ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14⁺CD16⁺ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393, increased CD14⁺CD16⁺ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14⁺CD16⁺ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14⁺CD16⁺ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14⁺CD16⁺ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14⁺CD16⁺ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14⁺CD16⁺ monocytes in dopamine rich brain regions.     

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background

Dopamine signaling is mediated by G_s protein-coupled “D₁-like” receptors (D₁ and D₅) and G_i-coupled “D₂-like” receptors (D_2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the G_i-coupled dopamine D₂ receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the G_s-coupled dopamine D₁-like receptor subtypes have never been identified on these cells. Activation of G_s-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D₁-like receptor is expressed on ASM, and modulates its function through G_s-coupling.

Methods

The mRNA and protein expression of dopamine D₁-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D₁ receptor, HASM cells were treated with dopamine or the dopamine D₁-like receptor agonists ({"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 or {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D₁ receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BK_Ca) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.

Results

Messenger RNA encoding the dopamine D₁ and D₅ receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D₁ receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D₁ receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D₁-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D₁-like receptor antagonists {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 or {"type":"entrez-protein","attrs":{"text":"SCH39166","term_id":"1052842517","term_text":"SCH39166"}}SCH39166. {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.

Conclusions

These results demonstrate that the dopamine D₁ receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.     

Dopamine D1 receptor-induced signaling through TrkB receptors in striatal neurons

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist SKF38393, we found that Trk neurotrophin receptors were activated in embryonic day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by SKF38393 was accompanied by the phosphorylation of several Trk signaling proteins, including phospholipase Cgamma, Akt, and MAPK. Biotinylation experiments followed by immunostaining by phospho-TrkB-specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca(2+). These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate the effects of dopamine upon striatal neurons.     

Arylbenzazepines Are Potent Modulators for the Delayed Rectifier K+ Channel: A Potential Mechanism for Their Neuroprotective Effects

(±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D₁-like dopamine receptor agonistic activity. The precise mechanism for the (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-mediated neuroprotection remains elusive. We report here that (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is a potent blocker for delayed rectifier K⁺ channel. (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 inhibited the delayed rectifier K⁺ current (I _K) dose-dependently in rat hippocampal neurons. The IC ₅₀ value for inhibition of I _K was 41.9±2.3 µM (Hill coefficient = 1.81±0.13, n = 6), whereas that for inhibition of I _A was 307.9±38.5 µM (Hill coefficient = 1.37±0.08, n = 6). Thus, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is 7.3-fold more potent in suppressing I _K than I _A. Moreover, the inhibition of I _K by (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 with the K⁺ channel. The intracellular application of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 had no effects of on I _K, indicating that the compound most likely acts at the outer mouth of the pore of K⁺ channel. We also tested the enantiomers of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, R-(+) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-201), and S-(−) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-202), as well as {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393; all these compounds inhibited I _K. However, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I _K , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959.     

Action of Dopaminergic Agents on the Impulse Activity of Sensorimotor Cortex Neurons of Cats Performing a Conditioned Motor Task

We demonstrate that dopamine itself, a selective dopamine D1 receptor agonist, SKF 38393, and a selective dopamine D2 receptor agonist, quinpirole, exert both facilitatory and suppressive effects on neuronal activity in the sensorimotor cortex of the cat, which is related to a conditioned movement. These effects are mediated by activation of dopamine receptors. Our data can be used for understanding the mechanisms underlying modulation of the excitability of central neurons during various behavioral events under the influence of dopamine.     

Electrophysiological Effects of SKF83959 on Hippocampal CA1 Pyramidal Neurons: Potential Mechanisms for the Drug's Neuroprotective Effects

Although the potent anti-parkinsonian action of the atypical D₁-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D₁ receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D₁, D₂, 5-HT_2A/2C receptors and α₁-adrenoceptor, nor by intracellular dialysis of GDP-β-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959. In voltage-clamp experiments, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D₁ receptor activation. Moreover, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 increased Ih current through a D₁ receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug''s neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson''s disease.     

SKF83959, an Agonist of Phosphatidylinositol-Linked D1-Like Receptors,Promotes ERK1/2 Activation and Cell Migration in Cultured Rat Astrocytes

Extracellular signal-regulated kinase 1/2 (ERK1/2) is a member of the mitogen-activated protein kinase family. It can mediate cell migration. Classical dopamine receptor-mediated ERK1/2 phosphorylation is widely studied in neurons. Here, we report that ERK1/2 phosphorylation is also modulated by putative phosphatidylinositol-linked D₁-like receptors in cultured rat astrocytes. 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine ({"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959), an agonist of the putative phosphatidylinositol-linked D₁-like receptors, was found to enhance ERK1/2 phosphorylation, which then promoted the migration of cultured astrocytes. The {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-induced ERK1/2 phosphorylation was found to be Ca²⁺-independent based on the following observations: i. chelating intracellular Ca²⁺ did not inhibit ERK1/2 phosphorylation and astrocyte migration; ii. blockage of the release of intracellular Ca²⁺ from the endoplasmic reticulum by an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor did not attenuate ERK1/2 phosphorylation. However, inhibition of phospholipase C (PLC), the upstream molecule of internal Ca²⁺ release, disabled {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959’s ability to elevate the level of ERK1/2 phosphorylation. Both non-selective protein kinase C (PKC) inhibitor and PKCδ selective inhibitor prevented ERK1/2 phosphorylation increase and astrocyte migration, but PKCα inhibitor did not. This suggests that Ca²⁺-independent and diacylglycerol-dependent PKCδ acts downstream of putative phosphatidylinositol-linked D₁-like receptor activation and mediates {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-induced elevation of ERK1/2 phosphorylation in order to modulate astrocyte migration. In conclusion, our results demonstrate that {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-induced increases in ERK1/2 phosphorylation and astrocyte migration are dependent on PLC-PKCδ signals. This might help us to further understand the functions of the putative phosphatidylinositol-linked D₁-like receptors in the nervous system.     

5-hydroxytryptamine has an endothelium-derived hyperpolarizing factor-like effect on coronary flow in isolated rat hearts

Background

5-hydroxytryptamine (5-HT)-induced coronary artery responses have both vasoconstriction and vasorelaxation components. The vasoconstrictive effects of 5-HT have been well studied while the mechanism(s) of how 5-HT causes relaxation of coronary arteries has been less investigated. In isolated rat hearts, 5-HT-induced coronary flow increases are partially resistant to the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME) and are blocked by 5-HT₇ receptor antagonists. In the present study, we investigated the role of 5-HT₇ receptor in 5-HT-induced coronary flow increases in isolated rat hearts in the absence of L-NAME, and we also evaluated the involvement of endothelium-derived hyperpolarizing factor (EDHF) in 5-HT-induced coronary flow increases in L-NAME-treated hearts with the inhibitors of arachidonic acid metabolism and the blockers of Ca²⁺-activated K⁺ channels.

Results

In isolated rat hearts, 5-HT and the 5-HT₇ receptor agonist 5-carboxamidotryptamine induced coronary flow increases, and both of these effects were blocked by the selective 5-HT₇ receptor antagonist SB269970; in SB269970-treated hearts, 5-HT induced coronary flow decreases, which effect was blocked by the 5-HT_2A receptor blocker {"type":"entrez-nucleotide","attrs":{"text":"R96544","term_id":"982204","term_text":"R96544"}}R96544. In L-NAME-treated hearts, 5-HT-induced coronary flow increases were blocked by the phospholipase A₂ inhibitor quinacrine and the cytochrome P450 inhibitor SKF525A, but were not inhibited by the cyclooxygenase inhibitor indomethacin. As to the effects of the Ca²⁺-activated K⁺ channel blockers, 5-HT-induced coronary flow increases in L-NAME-treated hearts were inhibited by TRAM-34 (intermediate-conductance Ca²⁺-activated K⁺ channel blocker) and UCL1684 (small-conductance Ca²⁺-activated K⁺ channel blocker), but effects of the large-conductance Ca²⁺-activated K⁺ channel blockers on 5-HT-induced coronary flow increases were various: penitrem A and paxilline did not significantly affect 5-HT-induced coronary flow responses while tetraethylammonium suppressed the coronary flow increases elicited by 5-HT.

Conclusion

In the present study, we found that 5-HT-induced coronary flow increases are mediated by the activation of 5-HT₇ receptor in rat hearts in the absence of L-NAME. Metabolites of cytochrome P450s, small-conductance Ca²⁺-activated K⁺ channel, and intermediate-conductance Ca²⁺-activated K⁺ channel are involved in 5-HT-induced coronary flow increases in L-NAME-treated hearts, which resemble the mechanisms of EDHF-induced vasorelaxation. The role of large-conductance Ca²⁺-activated K⁺ channel in 5-HT-induced coronary flow increases in L-NAME-treated hearts needs further investigation.     

Coexistence of inhibitory dopamine D-1 and excitatory D-2 receptors on the same caudate nucleus neurons

Microiontophoretic studies using cats anesthetized with alpha-chloralose were performed to determine whether or not dopamine D-1 and D-2 receptors co-exist in the same caudate nucleus (CN) neurons that receive inputs from the substantia nigra (SN), and in which spikes elicited by SN stimulation were blocked by domperidone, a selective D-2 antagonist. Iontophoretic application of dopamine produced a dose-dependent inhibition of spontaneous firing in 2 of 4 spontaneously active CN neurons and an increase in firing in the remaining 2 neurons. However, dopamine inhibited the glutamate-induced firing in 31 of 32 CN neurons that were not spontaneously active. Similar inhibition with iontophoretically applied SKF 38393, a selective D-1 agonist, was observed in 33 of 34 spontaneously inactive neurons tested. When the effects of dopamine, SKF 38393 and bromocriptine (D-2 agonist) were examined on the same CN neurons, the inhibitory effects of both dopamine and SKF 38393 were seen in 14 of 15 neurons, and both an inhibition by SKF 38393 and an excitation by bromocriptine were observed in 15 of 17 neurons. The inhibitory effects of dopamine and SKF 38393 were antagonized by haloperidol and SCH 23390 (D-1 antagonist) without being affected by domperidone. Furthermore, the dopamine-induced inhibition was converted to an excitation during simultaneous application of SCH 23390 in 6 of 10 CN neurons, and this excitation was antagonized by domperidone. These results strongly suggest that the inhibitory D-1 and excitatory D-2 receptors co-exist on the same CN neurons receiving inputs from the SN.     

B-HT 920 stimulates postsynaptic D2 dopamine receptors in the normal rat: electrophysiological and behavioral evidence

The putative autoreceptor-selective dopamine (DA) agonist B-HT 920 was tested using electrophysiological and behavioral models thought to reflect actions at postsynaptic D2 DA receptors. Direct iontophoretic application of B-HT 920 onto nucleus accumbens neurons caused a current-dependent inhibition of firing which could be attenuated by pretreatment with alpha-methyl-p-tyrosine (to deplete DA) and reinstated (enabled) by concurrent administration of the selective D1 DA receptor agonist SKF 38393. These findings suggest that, like other selective D2 DA receptor agonists, the postsynaptic effects of B-HT 920 require concurrent stimulation of D1 DA receptors. Behavioral indices of postsynaptic D2 DA receptor stimulation (stereotyped sniffing and rearing) were also evident following combined treatment with B-HT 920 and SKF 38393. Moreover, similar "low-level" stereotyped behaviors were also observed when B-HT 920 was administered alone following pretreatment with the alpha-2 adrenoceptor antagonists idazoxane and piperoxane, suggesting that alpha-2 agonist actions of B-HT 920, in some way, mask the expression of D2 receptor-mediated stereotyped responses. When B-HT 920 was combined with SKF 38393 following pretreatment with idazoxane, both the intensity and form (continual licking and gnawing) of stereotyped behavior was enhanced. Taken together, these electrophysiological and behavioral findings indicate that B-HT 920 possesses the properties of a selective D2 DA receptor agonist and cannot be considered as a DA autoreceptor-selective compound.     

c-Fos Expression by Dopaminergic Receptor Activation in Rat Hippocampal Neurons

While dopamine is likely to modulate hippocampal synaptic plasticity, there has been little information about how dopamine affects synaptic transmission in the hippocampus. The expression of IEGs including c-fos has been associated with late phase LTP in the CA1 region of the hippocampus. The induction of c-fos by dopaminergic receptor activation in the rat hippocampus was investigated by using semiquantitative RT-PCR and immuno-cytochemistry. The hippocampal slices which were not treated with dopamine showed little expression of c-fos mRNA. However, the induction of c-fos mRNA was detected as early as 5 min after dopamine treatment, peaked at 60 min, and remained elevated 5 h after treatment. Temporal profiles of increases in c-fos mRNA by R(+)-SKF-38393 (50 M) and forskolin (50 M) were similar to that of dopamine. An increase in [cAMP] was observed in dopamine-, SKF-, or forskolin-treated hippocampal slices. By immunocytochemical studies, control hippocampal cells showed little expression of c-Fos immunoreactivity. However, when cells were treated with dopamine, an increase in the expression of c-Fos immunoreactivity was observed after treatment for 2 h. The treatment of hippocampal neurons with R(+)-SKF38393 (50 M) or forskolin (50 M) also induced a significant increase in c-Fos expression. These results indicate that the dopamine D1 receptor-mediated cAMP dependant pathway is associated with the expression of c-Fos in the hippocampal neurons. These data are consistent with the possible role of endogenous dopamine on synaptic plasticity via the regulation of gene expression. Furthermore, these results imply that dopamine might control the process of memory storage in the hippocampus through gene expression.     

SKF38393抑制大鼠DRG分离神经元GABA-激活电流

在大鼠新鲜分离ＤＲＧ神经元标本上应用全细胞膜片箝记录,观察了多巴胺Ｄ１受体的选择性激动剂ＳＫＦ３８３９３ＨＣＩ对ＧＡＢＡ－激活电流的作用。大部分受检细胞对ＧＡＢＡ敏感,１０＾－６－１０＾－３－ｍｏｌ／Ｌ ＧＡＢＡ可于引起呈剂量依赖性的明显去敏感作用的内向电流。     

Alteration of striatal dopaminergic neurotransmission in a mouse model of DYT11 myoclonus-dystonia

Background

DYT11 myoclonus-dystonia (M-D) syndrome is a neurological movement disorder characterized by myoclonic jerks and dystonic postures or movement that can be alleviated by alcohol. It is caused by mutations in SGCE encoding ε-sarcoglycan (ε-SG); the mouse homolog of this gene is Sgce. Paternally-inherited Sgce heterozygous knockout (Sgce KO) mice exhibit myoclonus, motor impairment and anxiety- and depression-like behaviors, modeling several clinical symptoms observed in DYT11 M-D patients. The behavioral deficits are accompanied by abnormally high levels of dopamine and its metabolites in the striatum of Sgce KO mice. Neuroimaging studies of DYT11 M-D patients show reduced dopamine D2 receptor (D2R) availability, although the possibility of increased endogenous dopamine, and consequently, competitive D2R occupancy cannot be ruled out.

Methodology/Principal Findings

The protein levels of striatal D2R, dopamine transporter (DAT), and dopamine D1 receptor (D1R) in Sgce KO mice were analyzed by Western blot. The striatal dopamine release after amphetamine injection in Sgce KO mice were analyzed by microdialysis in vivo. The striatal D2R was significantly decreased in Sgce KO mice without altering DAT and D1R. Sgce KO mice also exhibited a significant increase of dopamine release after amphetamine injection in comparison to wild-type (WT) littermates.

Conclusion/Significance

The results suggest ε-SG may have a role in the regulation of D2R expression. The loss of ε-SG results in decreased striatal D2R, and subsequently leads to increased discharge of dopamine which could contribute to the behavioral impairment observed in DYT11 dystonia patients and in Sgce KO mice. The results suggest that reduction of striatal D2R and enhanced striatal dopamine release may contribute to the pathophysiology of DYT11 M-D patients.     

Progestin facilitation of lordosis in rodents involves adenylyl cyclase activity in the ventral tegmental area

Increasing cAMP, or activating dopamine type 1 (D(1)) or GABA(A)/benzodiazepine receptor complexes (GBRs), in the ventral tegmental area (VTA) enhances lordosis of rodents. Whether D(1)- and/or GBR-mediated increases in progestin-facilitated lordosis involve the cAMP-synthesizing enzyme, adenylyl cyclase, in the VTA, was investigated. In Experiment 1, ovariectomized estradiol (E(2); 10 microg at h 0)+progesterone (P; 250 microg at h 45)-primed hamsters first received bilateral infusions of the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA; 12 microM/side), or vehicle, and then were infused with the D(1) agonist, SKF38393 (100 ng/side), the GBR agonist, muscimol (100 ng/side), or vehicle, to the VTA. Lordosis was evaluated before and 30 min after each infusion. In Experiment 2, ovariectomized, E(2)-primed (10 microg at h 0) rats received VTA infusions of DDA (12 microM/side) or vehicle; SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle; and the neurosteroid, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP; 100 or 200 ng/side), or beta-cyclodextrin vehicle. Lordosis was assessed before the series of infusions, immediately after drug infusions and 10 or 60 min after 3alpha,5alpha-THP infusions. Progestin- or progestin plus SKF38393-or muscimol-mediated increases in lordosis were blocked by DDA pretreatment. Thus, in the VTA, progestins' membrane action may involve adenylyl cyclase.     

Angiotensin-2-Mediated Ca2+ Signaling in the Retinal Pigment Epithelium: Role of Angiotensin-Receptor- Associated-Protein and TRPV2 Channel

Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca²⁺inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca²⁺response. RPE cells from Atrap^−/− mice showed smaller AngII-evoked Ca²⁺peak (by 22%) and loss of sustained Ca²⁺elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca²⁺-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca²⁺-rise. Application of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca²⁺transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca²⁺transients in the RPE by releasing Ca²⁺from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca²⁺elevation.     

Role of Endogenous ENaC and TRP Channels in the Myogenic Response of Rat Posterior Cerebral Arteries

Aims

Mechanogated ion channels are predicted to mediate pressure-induced myogenic vasoconstriction in small resistance arteries. Recent findings have indicated that transient receptor potential (TRP) channels and epithelial sodium channels (ENaC) are involved in mechanotransduction. The purpose of this study was to investigate the role of TRP channels and ENaC in the myogenic response. Our previous study suggested that ENaC could be a component of the mechanosensitive ion channels in rat posterior cerebral arteries (PCA). However, the specific ion channel proteins mediating myogenic constriction are unknown. Here we found, for the first time, that ENaC interacted with TRPM4 but not with TRPC6 using immunoprecipitation and confocal microscopy.

Methods and Results

Treatment with a specific βENaC inhibitor, amiloride, a specific TRPM4 inhibitor, 9-phenanthrol, and a TRPC6 inhibitor, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365, resulted in inhibition of the pressure-induced myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with small interfering RNA of βENaC, TRPM4, and TRPC6. Co-treatment with amiloride and 9-phenanthrol showed a similar inhibitory effect on myogenic contraction compared to single treatment with amiloride or 9-phenanthrol. The myogenic response was not affected by 9-phenanthrol or amiloride treatment in PCA transfected with βENaC or TRPM4 siRNA, respectively. However, pressure-induced myogenic response was fully inhibited by co-treatment with amiloride, 9-phenanthrol, and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365, and by treatment with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 in PCA transfected with βENaC siRNA.

Conclusion

Our results suggest that ENaC, TRPM4, and TRPC6 play important roles in the pressure-induced myogenic response, and that ENaC and TRPM4 interact in rat PCA.