Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.     

Suppression of the morphine-induced rewarding effect in the rat with neuropathic pain: implication of the reduction in mu-opioid receptor functions in the ventral tegmental area

The present study was designed to investigate the rewarding effect, G-protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham-operated and sciatic nerve-ligated rats in the increases in guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N-propylnoraporphine or the D3 receptor agonist 7-hydroxy-2-dipropylaminotetralin (7-OH DPAT). In contrast, the increases in [35S]GTPgammaS binding to membranes of the VTA induced by either morphine or a selective micro -opioid receptor agonist [d-Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve-ligated rats as compared with sham- operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine-induced place preference under neuropathic pain may result from a decrease in the morphine-induced DA release in the N.Acc with reduction in the mu-opioid receptor-mediated G-protein activation in the VTA.     

Mechanisms of mu opioid receptor/G-protein desensitization in brain by chronic heroin administration

Previous studies have shown that chronic opiate treatment decreases mu opioid-stimulated [35S]GTPgammaS binding in specific brain regions. To extend these findings, the present study investigated DAMGO-stimulated [35S]GTPgammaS binding in membrane homogenates and coronal sections from rats non-contingently administered heroin. Rats were administered saline or increasing doses of heroin i.v. hourly up to 288 mg/kg/day over 40 days. In brain sections, chronic heroin administration decreased DAMGO-stimulated [35S]GTPgammaS binding in medial thalamus and amygdala, with no effect in cingulate cortex or nucleus accumbens. Chronic heroin administration also reduced [35S]GTPgammaS binding stimulated by the principal metabolite of heroin, 6-monoacetylmorphine. In contrast, no significant changes in mu opioid receptor binding were observed in amygdala or thalamus using [3H]DAMGO autoradiography. In membranes from amygdala and thalamus, chronic heroin treatment decreased the maximal effect of DAMGO in stimulating [35S]GTPgammaS binding, with no effect on DAMGO potency. GTPgammaS saturation analysis showed that chronic heroin treatment decreased the Bmax, and increased the K(D), of DAMGO-stimulated [35S]GTPgammaS binding. These data suggest potential mechanisms by which chronic agonist treatment produces opioid receptor/G-protein desensitization in brain.     

Orexin A in the ventral tegmental area induces conditioned place preference in a dose-dependent manner: Involvement of D1/D2 receptors in the nucleus accumbens

It has been shown that orexin A in the ventral tegmental area (VTA) is necessary for development of morphine place preference. Additionally, D1 and D2 dopamine receptors in the nucleus accumbens (NAc) have critical roles in motivation and reward. However, little is known about the function of orexin in conditioned place preference (CPP) in rats and involvement of D1/D2 receptors in the NAc. In the present study, we investigated the effect of direct administration of orexin A into the VTA, and examined the role of intra-accumbal dopamine receptors in development (acquisition) of reward-related behaviors in the rats. Adult male Wistar rats were unilaterally implanted by two separate cannulae into the VTA and NAc. The CPP paradigm was used, and, conditioning score and locomotor activity were recorded by Ethovision software. The results showed that unilateral intra-VTA administration of orexin A (27, 53 and 107ng/0.3μl saline) during conditioning phase induced CPP in a dose-dependent manner. The most effective dose of intra-VTA orexin-A in eliciting CPP was 107ng. However, intra-NAc administration of SCH 23390 (0.25, 1 and 4μg/0.5μl saline), a D1 receptor antagonist, and sulpiride (0.25, 1 and 4μg/0.5μl DMSO), a D2 receptor antagonist, inhibited the development of orexin-induced CPP. The inhibitory effect of D2 but not D1 receptor antagonist was exerted in a dose-dependent manner. It is supposed that the activation of VTA dopaminergic neuron by orexin impresses the D2 receptors more than D1 receptors in the NAc.     

Enhanced morphine withdrawal and micro -opioid receptor G-protein coupling in A2A adenosine receptor knockout mice

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.     

Repeated cocaine administration changes the function and subcellular distribution of adenosine A1 receptor in the rat nucleus accumbens

Adenosine A1 receptor (A1) protein and mRNA is increased in the nucleus accumbens following repeated cocaine treatment. In spite of this protein up-regulation, A1 agonist-stimulated [35S]GTPgammaS binding was attenuated in accumbens homogenates of rats withdrawn for 3 weeks from 1 week of daily cocaine injections. Cellular subfractionation revealed that the discrepancy between total A1 protein and G protein coupling resulted from a smaller proportion of receptors in the plasma membrane. The decrease in functional receptor in the plasma membrane was further indicated by diminished formation of heteromeric receptor complex consisting of A1 and dopamine D1A receptors. To explore the functional significance of the altered distribution of A1 receptors, at 3 weeks after discontinuing repeated cocaine or saline, animals were injected with cocaine and 45 min later the subcellular distribution of A1 receptors quantified. Whereas a cocaine challenge in repeated saline-treated animals induced a marked increase in membrane localization of the A1 receptor, the relative distribution of receptors in repeated cocaine rats was not affected by acute cocaine. These data suggest that the sorting and recycling of A1 receptors is dysregulated in the nucleus accumbens as the consequence of repeated cocaine administration.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain

Chronic treatment of rats with delta9-tetrahydrocannabinol (delta9-THC) results in tolerance to its acute behavioral effects. In a previous study, 21-day delta9-THC treatment in rats decreased cannabinoid activation of G proteins in brain, as measured by in vitro autoradiography of guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding. The present study investigated the time course of changes in cannabinoid-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding in both brain sections and membranes, following daily delta9-THC treatments for 3, 7, 14, and 21 days. Autoradiographic results showed time-dependent decreases in WIN 55212-2-stimulated [35S]GTPgammaS and [3H]WIN 55212-2 binding in cerebellum, hippocampus, caudate-putamen, and globus pallidus, with regional differences in the rate and magnitude of down-regulation and desensitization. Membrane binding assays in these regions showed qualitatively similar decreases in WIN 55212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding (using [3H]SR141716A), and demonstrated that decreases in ligand binding were due to decreases in maximal binding values, and not ligand affinities. These results demonstrated that chronic exposure to delta9-THC produced time-dependent and region-specific down-regulation and desensitization of brain cannabinoid receptors, which may represent underlying biochemical mechanisms of tolerance to cannabinoids.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

Preprodynorphin mediates locomotion and D2 dopamine and mu-opioid receptor changes induced by chronic 'binge' cocaine administration

Evidence suggests that the kappa-opioid receptor (KOP-r) system plays an important role in cocaine addiction. Indeed, cocaine induces endogenous KOP activity, which is a mechanism that opposes alterations in behaviour and brain function resulting from repeated cocaine use. In this study, we have examined the influence of deletion of preprodynorphin (ppDYN) on cocaine-induced behavioural effects and on hypothalamic-pituitary-adrenal axis activity. Furthermore, we have measured mu-opioid receptor (MOP-r) agonist-stimulated [(35)S]GTPgammaS, dopamine D(1), D(2) receptor and dopamine transporter (DAT) binding. Male wild-type (WT) and ppDYN knockout (KO) mice were injected with saline or cocaine (45 mg/kg/day) in a 'binge' administration paradigm for 14 days. Chronic cocaine produced an enhancement of locomotor sensitisation in KO. No genotype effect was found on stereotypy behaviour. Cocaine-enhanced MOP-r activation in WT but not in KO. There was an overall decrease in D(2) receptor binding in cocaine-treated KO but not in WT mice. No changes were observed in D(1) and DAT binding. Cocaine increased plasma corticosterone levels in WT but not in KO. The data confirms that the endogenous KOP system inhibits dopamine neurotransmission and that ppDYN may mediate the enhancement of MOP-r activity and the activation of the hypothalamic-pituitary-adrenal axis after chronic cocaine treatment.     

Regulation of dopamine D-receptor activation in vivo by protein phosphatase 2B (calcineurin)

Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal and D1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increased kinase-mediated phosphorylation.     

Mechanisms of ligand binding and efficacy at the human D2(short) dopamine receptor

Mechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2-dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPgammaS binding and to inhibit forskolin-stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2-dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute to binding affinity, potency and efficacy; and (iv) for the 2-dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.     

Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression

The roles of conserved aspartates in the third transmembrane domain of the rat mu opioid receptor (RMOR) were explored with mutations of D3.32(147) and D3.49(164). D3.49(164) in the highly conserved DRY motif was mutated to 13 amino acids. Except for the D3.49(164)E mutant, each mutant displayed little or no detectable [(3)H]diprenorphine binding, and pretreatment with naloxone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, and -E mutants were further studied. D3.32(147) was substituted with A or N. All seven mutants exhibited similar binding affinities for the antagonist [(3)H]diprenorphine as the wild-type. The D3.49(164)H, -Q, -Y, and -M mutants, but not the D3.49(164)E and D3.32(147) mutants, exhibited enhanced basal [(35)S]GTPgammaS binding which was comparable to the maximally activated level of the wild-type and was related to expression levels. Naloxone, naltrexone, and naloxone methiodide significantly inhibited the basal [(35)S]GTPgammaS binding of the D3.49(164) mutants, indicating inverse agonist activities. Treatment of the D3.49(164)Y mutant with pertussis toxin greatly reduced the basal [(35)S]GTPgammaS binding, demonstrating constitutive activation of Galpha(i)/Galpha(o). The D3.49(164)H, -Y, -M, and -Q mutants had higher affinities for DAMGO than the wild-type, which were not significantly lowered by GTPgammaS. Thus, mutation of D3.49(164) to H, Y, M, or Q in RMOR resulted in receptor assuming activated conformations. In contrast, the D3.49(164)E mutant displayed significantly lower basal [(35)S]GTPgammaS binding and reduced affinity for DAMGO. Upon incubation of membranes at 37 degrees C, the constitutively active D3.49(164)Y mutant was structurally less stable, whereas the inactivated D3.49(164)E mutant was more stable, than the wild-type. Computational simulations showed that the E3.49 side chain interacted strongly with the conserved R3.50 in the DRY motif and stabilized the inactive form of the receptor. Taken together, these results indicate that D3.49 plays an important role in constraining the receptor in inactive conformations.     

Constitutive activation of the delta opioid receptor by mutations in transmembrane domains III and VII.

We have investigated whether transmembrane amino acid residues Asp128 (domain III), Tyr129 (domain III) [corrected], and Tyr308 (domain VII) in the mouse delta opioid receptor play a role in receptor activation. To do so, we have used a [35S]GTPgammaS (where GTPgammaS is guanosine 5'-3-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [35S]GTPgammaS binding was increased for every mutant receptor under study (1.6-2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents alpha-aminobutyric acid) efficiently reduced basal [35S]GTPgammaS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [35S]GTPgammaS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp128 (III) and Tyr308 (VII) interact with each other and that Tyr129 (III) undergoes H bonding with His278 (VI). Thus, Asp128, Tyr129, and Tyr308 may be involved in a network of interhelical bonds, which contributes to maintain the delta receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.     

Alpha2-adrenergic agonist modulation of [35S]GTPgammaS binding to guanine-nucleotide-binding-proteins in human platelet membranes.

Alpha2-adrenoceptor (alpha2-AR)-regulated binding of the labelled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. Under optimal conditions, the potent alpha2-AR agonist, 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), increased the binding of [35S]GTPgammaS up to approximately 1.8 fold, with half-maximal increase at 60 nM and was competitively inhibited by the alpha2-AR antagonist Rauwolscine. The actions of both UK 14304 and Rauwolscine were modulated by monovalent and divalent cation levels, as well as by the concentrations of GDP. [35S]GTPgammaS binding induced by UK 14304 had a Kd value of 4.5 +/- 0.3 nM and a Bmax value of 4.15 +/- 0.40 pmol/mg protein. The rank order of potencies of adrenergic ligands tested in stimulating [3S]GTPgammaS binding and inhibiting forskolin-stimulated c-AMP accumulation was UK 14304> Guanabenz acetate> Oxymetazoline hydrochloride> B-HT 920 dihydrochloride> p-Aminoclonidine hydrochloride> Clonidine hydrochloride. The data presented indicate that enhancement of [35S]GTPgammaS binding by alpha2-AR in human platelet membranes provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of ligands at the alpha2-AR.     

Cav1.3 L-type Ca2+ channels mediate long-term adaptation in dopamine D2L-mediated GluA1 trafficking in the dorsal striatum following cocaine exposure

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesostriatal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization in mice we find that repeated cocaine results in a basal reduction of Ser 845 GluA1 and cell surface GluA1 levels in the dorsal striatum (dStr) following a protracted withdrawal period, an adaptation that is dependent on Ca_v1.3 channels but not those expressed in the VTA. We find that the basally-induced decrease in this phosphoprotein is the result of recruitment of the striatal dopamine D2 pathway, as evidenced by enhanced levels of D2 receptor (D2R) mRNA expression and D2R function as examined using the D2R antagonist, eticlopride, as well as alterations in the phosphorylation status of several downstream molecular targets of D2R’s, including CREB, DARPP-32, Akt and GSK3β. Taken together with our recently published findings examining similar phenomena in the nucleus accumbens (NAc), these results underscore the utilization of divergent molecular mechanisms in the dStr, in mediating cocaine-induced persistent behavioral changes.     

Cav1.3 L-type Ca2+ channels mediate long-term adaptation in dopamine D2L-mediated GluA1 trafficking in the dorsal striatum following cocaine exposure

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesostriatal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization in mice we find that repeated cocaine results in a basal reduction of Ser 845 GluA1 and cell surface GluA1 levels in the dorsal striatum (dStr) following a protracted withdrawal period, an adaptation that is dependent on Ca_v1.3 channels but not those expressed in the VTA. We find that the basally-induced decrease in this phosphoprotein is the result of recruitment of the striatal dopamine D2 pathway, as evidenced by enhanced levels of D2 receptor (D2R) mRNA expression and D2R function as examined using the D2R antagonist, eticlopride, as well as alterations in the phosphorylation status of several downstream molecular targets of D2R’s, including CREB, DARPP-32, Akt and GSK3β. Taken together with our recently published findings examining similar phenomena in the nucleus accumbens (NAc), these results underscore the utilization of divergent molecular mechanisms in the dStr, in mediating cocaine-induced persistent behavioral changes.     

Methods for the evaluation of drug action at the human melatonin receptor subtypes

NIH3T3 fibroblast cells transfected with the full-length coding regions of the mt1 and MT2 human melatonin receptors stably expressed the receptor, coupled to a pertussis-toxin-sensitive G protein and exhibiting high affinity for melatonin. Both mt1 and MT2 melatonin receptors mediated the incorporation of [35S]GTPgammaS into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]GTPgammaS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]GTPgammaS binding. Luzindole did not increase basal [35S]GTPgammaS binding but competitively inhibited melatonin-stimulated [35S]GTPgammaS binding, thus exhibiting antagonist action. Two other mt1 antagonists, 4P-PDOT and N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. For the first time, these findings show important differences in analogue intrinsic activity between the human mt1 and MT2 melatonin receptor subtypes.     

In Vitro Autoradiographic Visualization of Guanosine-5'-O-(3-[35S]Thio)triphosphate Binding Stimulated by Sphingosine 1-Phosphate and Lysophosphatidic Acid

Sphingosine 1-phosphate or lysophosphatidic acid activation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to G proteins was studied by in vitro autoradiography in rat and guinea pig brain. The highest stimulation of [35S]GTPgammaS binding by sphingosine 1-phosphate was observed in the molecular layer of the cerebellum. Marked stimulation was observed in most forebrain areas, including neocortex and striatum. With the exception of the substantia gelatinosa and nucleus of the solitary tract, sphingosine 1-phosphate-enhanced binding was weaker in the brainstem and spinal cord. Lysophosphatidic acid-enhanced labeling was only observed in white matter areas. The G protein inhibitor 5'-p-fluorosulfonylbenzoyl guanosine completely inhibited lysophosphatidic acid-enhanced [35S]GTPgammaS binding but only partially sphingosine 1-phosphate-enhanced binding. N-Ethylmaleimide abolished binding stimulated by both agonists. Sphingosine 1-phosphate enhanced labeling by another GTP analogue (beta,gamma-imido[8-3H]guanosine-5'-triphosphate) similarly to that of [35S]GTPgammaS. Lysophosphatidic acid stimulated [35S]GTPgammaS binding in the olfactory bulb, glia limitans, and cortical subventricular zone of 1-day-old rats, whereas enhanced labeling was not observed in the latter area of 5-day-old rats. Sphingosine 1-phosphate stimulated binding in the cortical and striatal subventricular zones and olfactory bulb in 1- and 5-day-old rats. In the absence of radioligand for sphingosine 1-phosphate and lysophosphatidic acid receptors, [35S]GTPgammaS autoradiography provides a unique opportunity to study the spatial distribution, ontogeny, and coupling properties of these receptors.