Dopamine D2 receptor inhibition of adenylyl cyclase is abolished by acute ethanol but restored after chronic ethanol exposure (tolerance).

Dopamine D2 (D2) receptors seem to mediate reinforcing responses to addicting drugs. A stably transfected NG108-15 cell line expressing the long form of the rat brain D2 receptor (D2L) was used to determine how ethanol modifies D2 receptor coupling to adenylyl cyclase. Activation of D2L receptors by the D2 receptor-specific agonist R-(-)-2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) inhibits both basal and receptor-stimulated cAMP production in these cells. Ethanol added acutely prevents D2L receptor inhibition of cAMP production. After chronic exposure to ethanol, however, D2L receptor coupling to adenylyl cyclase becomes tolerant to rechallenge with ethanol, i.e., ethanol no longer inhibits D2L receptor coupling and NPA inhibition of cAMP production is restored. Acute ethanol does not change NPA binding to D2 receptor in cell membranes but abolishes guanosine-5'-O-(3-thio)triphosphate induction of a lower-affinity state; chronic ethanol is without effect. The protein kinase A (PKA) inhibitor adenosine 3',5' cyclic monophosphorothioate, Rp-isomer, prevents acute ethanol inhibition of D2L receptor coupling. In contrast, the PKA activator adenosine 3',5' cyclic monophosphorothioate, Sp-isomer, reverses chronic ethanol-induced tolerance of D2L receptor coupling, restoring coupling to an ethanol-sensitive state. These results suggest that D2L receptor coupling to adenylyl cyclase via G(i) develops tolerance to ethanol inhibition, which appears to be influenced by PKA activity.     

Interaction with sigma(1) protein, but not N-methyl-D-aspartate receptor, is involved in the pharmacological activity of donepezil

In the present study, we examined the interaction of (+/-)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-d-aspartate (NMDA) and sigma(1) receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the l-glutamate or glycine binding sites. The low potency of donepezil (IC(50) = 0.7-3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the sigma(1) receptor with high affinity (K(i) = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5-8, 1999). Thus, we sought to determine whether an interaction with the sigma(1) receptor may occur in vivo under physiologically relevant conditions by evaluating the sigma(1) receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the sigma(1) receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the sigma(1) receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective sigma(1) receptor agonist on these behavioral responses, and an interaction of the drug with the sigma(1) receptor must be considered in its pharmacological actions.     

Modulation of dopamine release in the guinea-pig retina by G(i)- but not by G(s)- or G(q)-protein-coupled receptors

The modulation of dopamine release from the guinea-pig retina was studied using maximally effective concentrations of 10 agonists acting on G(i)-, G(s)- or G(q)-protein-coupled receptors (PCRs). Retinal discs were preincubated with [(3)H]noradrenaline and superfused; tritium overflow was evoked electrically. The following compounds acting on G(i)-PCRs reduced the tritium overflow, which represents quasi-physiological dopamine release under the experimental conditions of our study: the dopamine and alpha(2)-adrenoceptor agonist B-HT 920 by 95%, the muscarinic agonist oxotremorine by 96%, melatonin by 94%, the cannabinoid agonist WIN 55,212-2 by 71% and histamine by 66%. Tritium overflow was not affected by serotonin or by agonists acting on G(s)-PCRs (ACTH1-24 and the beta-adrenoceptor agonist procaterol) and G(q)-PCRs (angiotensin II and bradykinin). The effects of B-HT 920, oxotremorine and melatonin were studied in more detail using appropriate antagonists. The inhibitory effect of a submaximally active concentration of B-HT 920 was counteracted by the dopamine D(2/3) antagonist haloperidol but not affected by the alpha(2)-adrenoceptor antagonist phentolamine. The muscarinic antagonist atropine shifted to the right the concentration-response curve of oxotremorine (pA(2) 8.7) and the melatonin MT(2) antagonist 4-P-PDOT produced a rightward shift of the concentration-response curve of melatonin (pA(2) 10.6). Melatonin was also studied in superfused brain slices (from the guinea-pig) preincubated with [(3)H]noradrenaline. The electrically evoked tritium overflow in cerebrocortical, hippocampal and hypothalamic slices (representing quasi-physiological noradrenaline release) and in striatal slices (representing quasi-physiological dopamine release) was not affected by melatonin at a concentration that causes the maximum effect in retinal discs. In conclusion, dopamine release in the guinea-pig retina is inhibited via G(i)-PCRs including dopamine (D(2/3)), muscarinic and melatonin (MT(2)) receptors but not affected via any of the G(s)- or G(q)-PCRs under study. Unlike in the retina, melatonin fails to inhibit monoamine release in four brain regions of the guinea-pig.     

Differential effects of agonists on adenylyl cyclase superactivation mediated by the kappa opioid receptors: adenylyl cyclase superactivation is independent of agonist-induced phosphorylation, desensitization, internalization, and down-regulation

Prolonged activation of opioid receptors followed by agonist removal leads to adenylyl cyclase (AC) superactivation. In this study, we examined in CHO cells stably expressing the human or rat kappa opioid receptor (hkor or rkor) whether agonists had differential abilities to induce AC superactivation and whether the hkor and rkor exhibited differential AC superactivation. Pretreatment of the hkor with (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (U50,488H) induced AC superactivation in a time- and dose-dependent manner, reaching a plateau at 4 h and 0.1 microM. The extents of AC superactivation after a 4-h pretreatment of the hkor with saturating concentrations of agonists were in the order of the full agonists U50,488H, dynorphin A(1-17), (+/-)-ethylketocyclazocine, etorphine, and U69,593 > the high-efficacy partial agonist nalorphine > the low-efficacy partial agonists nalbuphine, morphine, and pentazocine. Interestingly, the full agonist levorphanol caused much lower AC superactivation than other full agonists and reduced the AC superactivation induced by U50,488H and dynorphin A(1-17) in a dose-dependent manner. The order of relative efficacies of agonists in causing AC superactivation mediated by the rkor was similar to that mediated by the hkor and the extents of AC superactivation were slightly lower. Because the rkor does not undergo U50,488H (1 microM)-induced phosphorylation, desensitization, internalization, and down-regulation in these cells, the degree of AC superactivation is independent of these processes. This is among the first reports to demonstrate that relative efficacies of agonists in causing AC superactivation generally correlated with those in activating G proteins and a full agonist reduced AC superactivation induced by another full agonist.     

P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo

The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An approximately 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 approximately 210-kD dimer and the approximately 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.     

Spinal 5-HT(2) and 5-HT(3) receptors mediate low, but not high, frequency TENS-induced antihyperalgesia in rats

Transcutaneous electrical nerve stimulation (TENS) is a form of non-pharmacological treatment for pain. Involvement of descending inhibitory systems is implicated in TENS-induced analgesia. In the present study, the roles of spinal 5-HT and alpha(2)-adrenoceptors in TENS analgesia were investigated in rats. Hyperalgesia was induced by inflaming the knee joint with 3% kaolin-carrageenan mixture and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The (1). alpha(2)-adrenergic antagonist yohimbine (30 microg), (2). 5-HT antagonist methysergide (5-HT(1). and 5-HT(2). 30 microg), one of the 5-HT receptor subtype antagonists, (3). NAN-190 (5-HT(1A), 15 microg), (4). ketanserin (5-HT(2A), 30 microg), (5). MDL-72222 (5-HT(3), 12 microg), or (6). vehicle was administered intrathecally prior to TENS treatment. Low (4 Hz) or high (100 Hz) frequency TENS at sensory intensity was then applied to the inflamed knee for 20 min and PWL was determined. Selectivity of the antagonists used was confirmed using respective agonists administered intrathecally. Yohimbine had no effect on the antihyperalgesia produced by low or high frequency TENS. Methysergide and MDL-72222 prevented the antihyperalgesia produced by low, but not high, frequency TENS. Ketanserin attenuated the antihyperalgesic effects of low frequency TENS whereas NAN-190 had no effect. The results from the present study show that spinal 5-HT receptors mediate low, but not high, frequency TENS-induced antihyperalgesia through activation of 5-HT(2A) and 5-HT(3) receptors in rats. Furthermore, spinal noradrenergic receptors are not involved in either low or high frequency TENS antihyperalgesia.     

The AcrAB-TolC pump is involved in macrolide resistance but not in telithromycin efflux in Enterobacter aerogenes and Escherichia coli

The role of the AcrAB-TolC pump in macrolide and ketolide susceptibility in Escherichia coli and Enterobacter aerogenes was studied. Efflux pump inhibitor restored erythromycin, clarithromycin, and telithromycin susceptibilities to multidrug-resistant isolates. No modification of telithromycin accumulation was detected in E. aerogenes acrAB or tolC derivatives compared to that in the parental strain. Two independent efflux pumps, inhibited by phenylalanine arginine beta-naphthylamide, expel macrolides and telithromycin in E. aerogenes.     

Vascular endothelium is critically involved in the hypotensive and hypovolemic actions of atrial natriuretic peptide

Atrial natriuretic peptide (ANP), via its vasodilating and diuretic effects, has an important physiological role in the maintenance of arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is highly expressed in vascular endothelium, but the functional relevance of this is controversial. To dissect the endothelium-mediated actions of ANP in vivo, we inactivated the GC-A gene selectively in endothelial cells by homologous loxP/Tie2-Cre-mediated recombination. Notably, despite full preservation of the direct vasodilating effects of ANP, mice with endothelium-restricted deletion of the GC-A gene (EC GC-A KO) exhibited significant arterial hypertension and cardiac hypertrophy. Echocardiographic and Doppler flow evaluations together with the Evan's blue dilution technique showed that the total plasma volume of EC GC-A KO mice was increased by 11-13%, even under conditions of normal dietary salt intake. Infusion of ANP caused immediate increases in hematocrit in control but not in EC GC-A KO mice, which indicated that ablation of endothelial GC-A completely prevented the acute contraction of intravascular volume produced by ANP. Furthermore, intravenous ANP acutely enhanced the rate of clearance of radio-iodinated albumin from the circulatory system in control but not in EC GC-A KO mice. We conclude that GC-A-mediated increases in endothelial permeability are critically involved in the hypovolemic, hypotensive actions of ANP.     

P-glycoprotein is not involved in the differential oral potency of naloxone and naltrexone

The poor oral bioavailability of the opioid receptor antagonist naloxone (NA) when compared with naltrexone (NX) may be related to a greater interaction of naloxone with the efflux drug transporter P-glycoprotein (P-gp). We studied the involvement of P-gp in the transepithelial transport of the two opioid receptor antagonists, using a validated human in vitro Caco-2 cell monolayer model. The bidirectional transport of NA and NX (1, 50 and 100 μ m ) across the monolayers was investigated in the presence and absence of the specific P-gp inhibitor GF120918 (4 μ m ). NA and NX showed equal transport rates between the apical-to-basolateral (A–B) and the basolateral-to-apical (B–A) directions and neither the influx nor the efflux transport was affected by the P-gp inhibitor ( P  > 0.05). In conclusion, NA and NX are not P-gp substrates. The differential oral bioavailability of the two opioid antagonists is P-gp independent.     

Both the antioxidant and D3 agonist actions of pramipexole mediate its neuroprotective actions in mesencephalic cultures

Pramipexole (PPX) is a full intrinsic activity, direct-acting dopamine (DA) agonist possessing 7-fold higher affinity for D3 than for D2 receptors. It also is a potent antioxidant. PPX was previously shown to be neuroprotective because it dose dependently attenuated the DA neuron loss produced by levodopa in mesencephalic cultures. Several different drugs with properties similar to PPX were studied here to better understand the mechanism or mechanisms responsible for this neuroprotective effect. The D3-preferring agonist 7-hydroxy-diphenylaminotetralin (7-OH-DPAT) and the D3 antagonist U99194, respectively, increased and decreased the neuroprotective effects of PPX in a dose-dependent fashion. Addition of the selective D2 agonist U95666 or the D2/D3 antagonists domperidone or raclopride did not affect PPX's neuroprotective effect. Interestingly, 7-OH-DPAT by itself did not attenuate the DA neuron loss produced by levodopa. However, when 7-OH-DPAT was combined with a low dose of the antioxidants U101033E or alpha-tocopherol, the toxic effects of levodopa were attenuated. Similar results were observed when the D3-preferring agonist PD128, 907 was studied. In addition, media conditioned by exposure of mesencephalic cultures incubated with all D3-preferring agonists studied was shown to enhance the growth of DA neurons in freshly harvested recipient cultures implicating a D3-mediated trophic activity in the neuroprotective effect. These data suggest that PPX's neuroprotective actions in the levodopa toxicity model are a consequence of its combined actions as a D3 receptor agonist and an antioxidant.     

Neuromedin N mimics the actions of neurotensin in the ventral tegmental area but not in the nucleus accumbens

Neuromedin N (NN) is a hexapeptide recently isolated from porcine spinal cord that shares a four-amino acid homology with the C-terminus of the biologically active tridecapeptide neurotensin (NT). Microinjection with NT into the ventral tegmental area or nucleus accumbens of rats has been shown to increase locomotor activity and dopamine (DA) metabolism in some limbic areas or to inhibit the motor stimulant effect of intra-accumbens administration of DA, respectively. In this study the effects of microinjected NN were compared with those of NT. After injection into the ventral tegmental area, NN was shown to be more potent than NT at increasing spontaneous motor activity and to produce an increase in DA metabolism in the nucleus accumbens, prefrontal cortex, diagonal band of Broca and septum. However, when injected into the nucleus accumbens, NN was markedly less potent than NT at inhibiting DA-induced behavioral hyperactivity. In addition to DA-related effects, i.c.v. injection with NT causes hypothermia, and i.c.v. administration with NN was without effect on colonic temperature. These data demonstrate that NN has a behavioral profile distinct from that of NT.     

Influence of chronic lithium treatment upon the motivational effects of opioids: alteration in the effects of mu- but not kappa-opioid receptor ligands.

The influence of chronic lithium (Li) treatment upon the secondary reinforcing effects of opioid agonists and antagonists was examined by use of an unbiased place preference conditioning procedure. Administration of the mu-agonist morphine to control rats resulted in marked preferences for the drug-associated place and a similar effect was observed in response to the psychostimulant d-amphetamine. In contrast, the selective kappa-opioid agonist U-69593 [(5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-1(pyrrolidinyl-1-oxaspirol(4,5) dec-8-yl benzeneacetamide)] and the opioid antagonist naloxone produced dose-related place aversions. Chronic administration of a Li-containing diet, which produced serum levels of 0.56 mmol/l, abolished the place preferences induced by morphine but not d-amphetamine. This treatment abolished the aversive effects of naloxone but did not modify those produced by U-69593. These data and those from a previous place conditioning study indicate that Li can function as an antagonist of mu-opioid receptor ligands in vivo and that this action underlies its motivational effects. Furthermore, the inability of chronic Li treatment to modify either the content or basal release of beta-endorphin in various brain regions suggests that this antagonism is mediated directly at the level of the opioid receptor and/or its transducer systems.     

Sirolimus oral absorption in rats is increased by ketoconazole but is not affected by D-alpha-tocopheryl poly(ethylene glycol 1000) succinate

The contributions of cytochrome P450 3A (CYP3A) and P-glycoprotein to sirolimus oral bioavailability in rats were evaluated by coadministration of sirolimus (Rapamune) with the CYP3A inhibitor ketoconazole or the P-glycoprotein inhibitor D-alpha-tocopheryl poly(ethylene glycol 1000) succinate (TPGS). Groups of six male Sprague-Dawley rats (250-300 g) were administered Rapamune (1 mg/kg) by oral gavage, alone and with ketoconazole (30 mg/kg) or TPGS (50 mg/kg). Sirolimus levels were measured in whole blood over a 6-h time course. Sirolimus C(max) (6.6 +/- 1.6 versus 26 +/- 7 ng/ml) and area under the concentration versus time curve from 0 to 6 h (AUC(0-6)) (22 +/- 7 versus 105 +/- 27 ng. h/ml) were increased 3- to 5-fold by ketoconazole. Median T(max) (1.5-2 h) was unchanged. TPGS had no effect on sirolimus absorption. The interaction of sirolimus with P-glycoprotein was also evaluated in vitro using HCT-8 and Caco-2 cell monolayers. Consistent with published reports, sirolimus was a good inhibitor of P-glycoprotein, inhibiting polarized basolateral-to-apical flux of rhodamine 123 with an IC(50) of 0.625 to 1.25 microM (cyclosporine caused >80% inhibition at 5 microM). Sirolimus did not demonstrate significant polarized flux in either direction using the same monolayers (basolateral-to-apical flux was <2 times the apical-to-basolateral). Moreover, sirolimus flux was not impacted by cyclosporine, suggesting that it does not undergo P-glycoprotein-mediated transport in this system. The lack of significant sirolimus transport by P-glycoprotein may, in part, explain the lack of a TPGS effect on sirolimus absorption in rats.     

Octreotide is not effective in the acute treatment of migraine

The aim of this study was to determine whether subcutaneous octreotide is effective for the treatment of acute migraine. Patients with migraine with and without aura as classified by the International Headache Society were recruited to a double-blind placebo-controlled crossover study. Patients were instructed to treat two attacks of at least moderate pain severity, with at least a 7 day interval, using subcutaneous 100 microg octreotide or matching placebo. The primary endpoint was the headache response defined as: severe or moderate pain becomes mild or nil, at 2 h. The primary endpoint was analysed using a Multilevel Analysis approach. Secondary end-points included associated symptoms and a four-point functional disability score. The study was powered to detect a 30% difference at an alpha of 0.05 and a beta of 0.8. A total of 51 patients were recruited, of whom 42 provided efficacy data on an attack treated with octreotide and 41 with placebo. Modelling the headache response as a binomial determined by treatment, using the patient as the level 2 variable, and considering a possible period effect, and sex and migraine type as other variables of interest, subcutaneous octreotide was not significantly superior to placebo. The two hour headache response rates were 20% for placebo and 14% for octreotide, whilst the two hour pain free rates were 7% and 2%, respectively. Subcutaneous octreotide 100 microg is not effective in the acute treatment of migraine when compared to placebo.     

5-hydroxytryptamine (HT)1A receptors and the tail-flick response. III. Structurally diverse 5-HT1A partial agonists attenuate mu- but not kappa-opioid antinociception in mice and rats.

This study examined the effects of structurally diverse 5-hydroxytryptamine (HT)1A partial agonists upon opioid-induced antinociception against noxious heat and pressure stimuli in rats and mice. The pyrimidinylpiperazines, buspirone, ipsapirone and gepirone, the halogenated phenylpiperazine, LY 165, 163 [1-(2-(4-aminophenyl)ethyl-4-(3-trifluoromethylphenyl)-piperazine], the heterobicylic arylpiperazine, (+/-)-flexinoxan, and the benzodiaxane, MDL 728328-[(4-(1,4-benzodioxon-2-ylmethylamino)butyl-8-azasp iro-(4,5)-decane-7,9-dione], exerted little or no effect upon basal latencies. In both mice and rats, each dose-dependently attenuated the antinociceptive action of the mu-opioid, morphine, against heat and pressure. In their presence, the morphine dose-response curve was shifted in parallel to the right with no loss of maximal effect. In mice, Schild analysis of the action of ipsapirone and gepirone yielded slopes of close to -1. In contrast to the partial agonists, the buspirone metabolite, 1-pyrimidinylpiperazine, which lacks 5-HT1A affinity, and the putative 5-HT1A antagonists, methiothepin, spiperone, BMY 7378 [(8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol [4]-decane-7,9-dione) 2HCl] and alprenolol, did not reduce the action of morphine. In rats, the antagonistic effect of buspirone, gepirone and ipsapirone could be blocked by BMY 7378. The 5-HT1A partial agonists also antagonized the antinociception-induced by the mu-opioid, sufentanil, but were virtually inactive against the selective kappa-opioid agonists, U69,593 (5 alpha,7 alpha,8 beta-(+)-N-methyl-N-[7-(l-pyrrolidinyl)-1-oxaspirol-(4,5)-dec-8-yl ] benzene-acetamide) and U50,488H (trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide methane sulfonate hydrate.(ABSTRACT TRUNCATED AT 250 WORDS)