Plasticity of first-order sensory synapses: interactions between homosynaptic long-term potentiation and heterosynaptically evoked dopaminergic potentiation.
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Persistent potentiations of the chemical and electrotonic components of the eighth nerve (NVIII) EPSP recorded in vivo in the goldfish reticulospinal neuron, the Mauthner cell, can be evoked by afferent tetanization or local dendritic application of an endogenous transmitter, dopamine (3-hydroxytyramine). These modifications are attributable to the activation of distinct intracellular kinase cascades. Although dopamine-evoked potentiation (DEP) is mediated by the cAMP-dependent protein kinase (PKA), tetanization most likely activates a Ca2+-dependent protein kinase via an increased intracellular Ca2+ concentration. We present evidence that the eighth nerve tetanus that induces LTP does not act by triggering dopamine release, because it is evoked in the presence of a broad spectrum of dopamine antagonists. To test for interactions between these pathways, we applied the potentiating paradigms sequentially. When dopamine was applied first, tetanization produced additional potentiation of the mixed synaptic response, but when the sequence was reversed, DEP was occluded, indicating that the synapses potentiated by the two procedures belong to the same or overlapping populations. Experiments were conducted to determine interactions between the underlying regulatory mechanisms and the level of their convergence. Inhibiting PKA does not impede tetanus-induced LTP, and chelating postsynaptic Ca2+ with BAPTA does not block DEP, indicating that the initial steps of the induction processes are independent. Pharmacological and voltage-clamp analyses indicate that the two pathways converge on functional AMPA/kainate receptors for the chemically mediated EPSP and gap junctions for the electrotonic component or at intermediaries common to both pathways. A cellular model incorporating these interactions is proposed on the basis of differential modulation of synaptic responses via receptor-protein phosphorylation. (+info)
Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.
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We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway. (+info)
Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone. Evidence for a third drug-binding site.
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P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport. (+info)
Zinc modulates antagonist interactions with D2-like dopamine receptors through distinct molecular mechanisms.
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Recently, zinc has been shown to modulate antagonist drug interactions with the D1 dopamine receptor (Schetz and Sibley, 1997) and the dopamine transporter (Norregaard et al., 1998). We now demonstrate that zinc also reversibly and dose-dependently modulates the specific binding of the butyrophenone antagonist [3H]methylspiperone to all D2-like dopamine receptors: D2L, D3, and D4. The molecular mechanisms of zinc regulation of these D2-like receptor subtypes are distinct because zinc inhibition of [3H]methylspiperone binding to the D4 receptor is noncompetitive by both equilibrium and kinetic measures (lower Bmax and essentially no change in koff), whereas the corresponding inhibition of zinc at D2L and D3 receptors is primarily characterized by competitive allosterism (increases in KD and koff). Interestingly, thermodynamic measurements reveal that the macroscopic properties of zinc binding are entropy-driven for all receptor subtypes, despite their having distinct molecular mechanisms. Zinc also reduces the binding affinity of the D2L receptor for [3H]raclopride, a structurally different antagonist of the substituted benzamide class. Sodium ions negatively modulate zinc inhibition of both sodium-insensitive [3H]methylspiperone binding and sodium-sensitive [3H]raclopride binding. In addition to its demonstrated effects on antagonist binding in membrane preparations, zinc also retards the functional effects of antagonist at the D2L receptor in intact cells. These findings suggest that synaptic zinc may be a factor influencing the effectiveness of therapies that rely on dopamine receptor antagonists. (+info)
Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons.
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1. The effect of serotonin (5-HT) on the hyperpolarization-activated cation current (IH) was studied in small-, medium- and large-diameter acutely isolated rat dorsal root ganglion (DRG) cells, including cells categorized as type 1, 2, 3 and 4 based on membrane properties. 5-HT increased IH in 91 % of medium-diameter DRG cells (including type 4) and in 67 % of large-diameter DRG cells, but not other DRG cell types. 2. The increase of IH by 5-HT was antagonized by spiperone but not cyanopindolol, and was mimicked by 5-carboxyamidotryptamine, but not (+)-8-hydroxydipropylaminotetralin (8-OH-DPAT) or cyanopindolol. These data suggested the involvement of 5-HT7 receptors, which were shown to be expressed by medium-diameter DRG cells using RT-PCR analysis. 3. 5-HT shifted the conductance-voltage relationship of IH by +6 mV without changing peak conductance. The effects of 5-HT on IH were mimicked and occluded by forskolin, but not by inactive 1,9-dideoxy forskolin. 4. At holding potentials negative to -50 mV, 5-HT increased steady-state inward current and instantaneous membrane conductance (fast current). The 5-HT-induced inward current and fast current were blocked by Cs+ but not Ba2+ and reversed at -23 mV, consistent with the properties of tonically activated IH. 5. In medium-diameter neurons recorded from in the current clamp mode, 5-HT depolarized the resting membrane potential, decreased input resistance and facilitated action potential generation by anode-break excitation. 6. The above data suggest that in distinct subpopulations of DRG neurons, 5-HT increases cAMP levels via activation of 5-HT7 receptors, which shifts the voltage dependence of IH to more depolarized potentials and increases neuronal excitability. (+info)
Pharmacological evidence for the 5-HT7 receptor mediating smooth muscle relaxation in canine cerebral arteries.
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1. We investigated in the present study whether 5-HT is able to exert direct relaxant responses in canine basilar and middle cerebral arteries via the 5-HT7 receptor. 2. In arterial rings deprived of endothelium and pre-contracted with prostaglandin F2 alpha (2 microM), 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine, sumatriptan or alpha-methyl-5-HT produced further increase in tone and/or slight relaxation. Blockade of 5-HT1B 1D and 5-HT2A receptors with GR127935 (1 microM) and ketanserin (0.1 microM), respectively, antagonized the vasoconstrictor component of the response and unmasked a concentration-dependent relaxation to 5-HT, 5-CT and 5-methoxytryptamine; sumatriptan and alpha-methyl-5-HT remained inactive as relaxant agonists. The rank order of agonist potency in both arteries was 5-CT > 5-HT > 5-methoxytryptamine >> sumatriptan > or = alpha-methyl-5-HT. 3. In dog basilar artery, pre-incubated with GR127935 (1 microM) and ketanserin (0.1 microM) and precontracted with prostaglandin F2 alpha (2 microM), the 5-HT7 ligands, clozapine (1 microM), mesulergine (0.3 microM), methiothepin (3 nM), risperidone (3 nM), spiperone (1 microM) and LY215840 (10-100 nM), produced significant rightward shifts of the concentration-response curves for 5-HT and 5-CT. Only methiothepin and risperidone reduced significantly the maximum relaxant response (Emax), whilst the other drugs behaved as competitive antagonists with affinity values (pKB) that significantly correlated with their binding affinity (pKi) at recombinant 5-HT7 receptors. 4. These data disclosing the involvement of the 5-HT7 receptor in cerebrovascular relaxation may be strongly relevant in the light of: (1) the involvement of 5-HT in migraine; (2) the putative linkage between cephalovascular vasodilatation and migraine headache; and (3) the relatively high 5-HT7 receptor affinity of migraine prophylactic 5-HT antagonists. (+info)
Effect of tetrahydropalmatine analogs on Fos expression induced by formalin-pain.
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AIM: To study the effect of tetrahydropalmatine (THP) analogs on Fos protein expression induced by formalin-pain and elucidate analgesic mechanism of THP analogs. METHODS: The pain response to Sprague Dawley rats was induced with formalin injected s.c. into the plantar surface of the right hindpaw. Fos protein expression in brain and spinal cord was investigated with immunohistochemistry. The numbers of Fos-like immunoreactive (FLI) neurons were counted with Leica Q570 image analyzer. RESULTS: In the groups of THP analogs and D2 antagonist spiperone, FLI neurons induced by intraperitoneal (i.p.) injection of THP analogs and spiperone were mainly located in the striatum and accumbens nucleus, and a few FLI neurons were also in sensorimotor cortex. In the D1 antagonist, D1 agonist, D2 agonist, saline and vehicle groups, FLI neurons were seldom seen in the striatum and accumbens nucleus. Moreover, the Fos protein expression induced by l-THP and spiperone could be prevented by the pre-treatment of the D2 agonist quinpirole but not D1 agonist SKF38393. In the formalin-pain group, FLI neurons were mainly distributed in ascending pain afferent system (APAS) and descending pain modulation system (DPMS). Following i.p. THP analogs, however, the numbers of FLI neurons induced by formalin-pain in the APAS, such as dorsal horn (mainly laminae I, II, IV-VI) were markedly decreased, while the numbers of FLI neurons in the DPMS, such as periaqueductal gray (PAG) and reticular paragigantocellular lateral nucleus (RPLN) were significantly increased. CONCLUSION: THP analogs enhanced the activity of brainstem DPMS by the blockade of D2 receptors in the striatum and accumbens nucleus, and sequentially inhibited the inputs of peripheral pain afferent message in spinal cord level. (+info)
Dihydroetorphine-induced place preference was mediated by dopamine D1 receptors in rats.
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AIM: To study the influence of dopamine (DA) receptor antagonists upon the rewarding property of dihydroetorphine (DHE). METHODS: Conditioned place preference (CPP) paradigm was used to characterize the rewarding effect of DHE. DA receptor antagonists were injected administered subcutaneously or peritoneally and microinjected into nucleus accumbens (NAcc). RESULTS: DHE (0.05, 0.5, and 5.0 micrograms.kg-1, s.c.) produced place preference (P < 0.01). Both the DA receptor antagonist haloperidol and the selective D1 receptor antagonist Sch-23390 attenuated the place preference produced by DHE (0.5 microgram.kg-1, s.c.). l-Sulpiride and spiperone, selective D2 receptor antagonists, had no such effects. CONCLUSION: The D1 (but not D2) receptors in NAcc are crucial in the mediation of the rewarding effect of DHE. (+info)