Increase In Number Of Receptors Research Articles

GABA-B receptors enhance GABA-A receptor currents by modulation of membrane trafficking in dentate gyrus granule cells

Activation of postsynaptic GABA-B receptors enhances tonic inhibition mediated by high-affinity extrasynaptic GABAA receptors in dentate gyrus granule cells (DGGCs), thalamocortical neurons, and cerebellar granule cells. We investigated the mechanism(s) of GABA current modulation by GABAB receptors in DGGCs using a combination of electrophysiological and biochemical approaches. In acute hippocampal brain slices the GABAB receptor agonist baclofen increased GABA-evoked currents in ∼2/3rds of DGGCs, significantly increasing GABAA currents by 41% on average. Nonstationary noise analysis was performed to estimate the effects of baclofen on single channel conductance, mean open time, and channel number; these estimates suggest that GABAB receptor activation increases receptor number but does not modify single channel properties of GABAA receptors. To directly assess baclofen-induced changes in plasma membrane expression of GABAA receptors, biotinylated western blots were performed. Treatment of hippocampal slices with baclofen significantly increased the surface expression of GABAA receptor subunits (both δ and γ2 subunits) and this effect was inhibited by the GABAB receptor antagonist CGP55845. These data indicate that changes in membrane trafficking and increased number of GABAA receptors in plasma membrane contribute to the enhancement of GABA currents produced by GABAB receptor activation in DGGCs.

Comparison of common platelet receptors between the chacma baboon (Papio ursinus) and human for use in pre-clinical human-targeted anti-platelet studies

Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.

Discovering the mechanisms underlying serotonin (5-HT)2A and 5-HT2C receptor regulation following nicotine withdrawal in rats.

We have previously demonstrated that nicotine withdrawal produces depression-like behavior and that serotonin (5-HT)2A/2C receptor ligands modulate that mood-like state. In the present study we aimed to identify the mechanisms (changes in radioligand binding, transcription or RNA-editing) related to such a behavioral outcome. Rats received vehicle or nicotine (0.4 mg/kg, s.c.) for 5 days in home cages. Brain 5-HT2A/2C receptors were analyzed on day 3 of nicotine withdrawal. Nicotine withdrawal increased [(3)H]ketanserin binding to 5-HT2A receptors in the ventral tegmental area and ventral dentate gyrus, yet decreased binding in the nucleus accumbens shell. Reduction in [(3)H]mesulergine binding to 5-HT2C receptors was seen in the ventral dentate gyrus. Profound decrease in the 5-HT2A receptor transcript level was noted in the hippocampus and ventral tegmental area. Out of five 5-HT2C receptor mRNA editing sites, deep sequencing data showed a reduction in editing at the E site and a trend toward reduction at the C site in the hippocampus. In the ventral tegmental area, a reduction for the frequency of CD 5-HT2C receptor transcript was seen. These results show that the reduction in the 5-HT2A receptor transcript level may be an auto-regulatory response to the increased receptor density in the hippocampus and ventral tegmental area during nicotine withdrawal, while decreased 5-HT2C receptor mRNA editing may explain the reduction in receptor labeling in the hippocampus. Serotonin (5-HT)2A/2C receptor ligands alleviate depression-like state in nicotine-withdrawn rats. Here, we show that the reduction in 5-HT2A receptor transcript level may be an auto-regulatory response to the increased receptor number in the hippocampus and ventral tegmental area during nicotine withdrawal, while attenuated 5-HT2C receptor mRNA editing in the hippocampus might explain reduced inverse agonist binding to 5-HT2C receptor and suggest a shift toward a population of more active receptors. 5-HT, serotonin; 5-HT2A R, 5-HT2A receptor; 5-HT2C R, 5-HT2C receptor.

Ultra-low exposure to alpha-7 nicotinic acetylcholine receptor partial agonists elicits an improvement in cognition that corresponds with an increase in alpha-7 receptor expression in rodents: implications for low dose clinical efficacy

Αlpha-7 neuronal nicotinic receptors (NNRs) are considered targets for cognitive enhancement in schizophrenia and Alzheimer's disease. AZD0328 is an alpha-7 NNR partial agonist that enhances cognition in rodents and nonhuman primates at sub-microgram to microgram doses. We hypothesized that increased expression of the alpha-7 receptor contributes to this beneficial activity at low doses and tested this by examining the effect of AZD0328 using in vivo and ex vivo binding, RT-PCR and cognitive function in rodents. AZD0328 (0.00178 mg/kg) was subcutaneously administered to mice 4, 24, 48 and 72 hours prior to testing in novel object recognition and produced a significant increase in cognition at 4, 24 and 48 h post-dosing. In vivo binding was examined in rat brain using [(3)H]AZ11637326 and there was a dose-dependent reduction in receptor binding at higher doses of AZD0328 (0.001-3 mg/kg), and a second alpha-7 partial agonist, SSR180711 (0.01-30 mg/kg). Lower doses of both compounds (0.0001 mg/kg) produced a significant increase in binding of [(3)H]AZ11637326. Ex vivo binding using [(125)I]-α-bungarotoxin, showed a significant increase in receptor number (B(max.)) in the frontal cortex or hippocampus with no significant effect on receptor affinity (K(d)) 2 h post administration of AZD0328. [(3)H]AZ11637326 administered 1.5 h following AZD0328 produced a significant increase in specific binding in rat brain regions. We found that the effect on receptor number was long-lasting, with [(125)I]-α-bungarotoxin binding increased in rats given AZD0328 for 2-48 h, but this was not accompanied by increased mRNA synthesis. SSR180711 produced a similar increase in B(max.) and specific binding with no effect on K(d). Therefore, trace dose of alpha-7 partial agonists has rapid onset and produces a profound, sustained effect on novel object recognition in mice that corresponds by dose to an increase in receptor number in rat brain. These findings provide an explanation for the acute and sustained benefit of alpha-7 receptor activation in working memory in nonhuman primates and guidance for drug development initiatives and treatment regimens for nicotinic partial agonists.

Calculated and actual changes in beta-adrenoceptor number associated with increases in rat and guinea-pig cardiac sensitivity.

Chronic catecholamine depletion induced by reserpine pretreatment of rats, or 6-hydroxydopamine pretreatment of guinea-pigs, resulted in an enhanced sensitivity of isolated papillary muscles to isoprenaline. This hypersensitivity was accompanied by 1.41-(rats) and 1.52-fold (guinea-pigs) increases in the number of [3H]dihydroalprenolol binding sites, without changes in binding affinity. An equation was derived for calculation of increases in receptor number. Application of this showed that substantially greater increases in receptor number were required (2.32- to 4.04-fold) to account for the degree of supersensitivity observed.

Sympathectomy does not cause supersensitivity to noradrenaline in rat prostate

Chemical sympathectomy classically produces a denervation supersensitivity assumed to be due to a general increase in receptor number on smooth muscle. However, we have suggested that denervation results rather in a spread of synaptic alpha1D‐adrenoceptors, resulting in an increased proportion of this subtype. We now examine whether these effects of sympathectomy are dependent on the prior presence of innervated alpha1D‐adrenoceptors by studying rat prostate in which innervated receptors are reported to be alpha1A. Animals were sympathectomised by 6‐hydroxydopamine injection. Contractions to nerve stimulation in prostate were markedly reduced by the alpha1A‐ antagonist RS100329, which was more potent than the alpha1D‐antagonist BMY 7378. Norepinephrine (NE) potency at producing contractions was unchanged by sympathectomy, so that denervation supersensitivity did not occur. RS100329 significantly shifted the potency of NE to a similar extent in tissues from vehicle‐treated and sympathectomised animals. BMY 7378 revealed a small alpha1D‐adrenoceptor component to NE contractions in sympathectomised but not control. Since supersensitivity does not occur in prostate, a tissue without a significant amount of alpha1D‐adrenoceptors, this may imply that the presence of functionally important innervated alpha1D‐adrenoceptors is required for denervation supersensitivity.

The Integrity of the Glycine Co-agonist Binding Site of N-Methyl-d-aspartate Receptors Is a Functional Quality Control Checkpoint for Cell Surface Delivery

N-Methyl-D-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an approximately 3 x 10(4) decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.

Enhanced [3H] Glutamate Binding in the Cerebellum of Insulin-Induced Hypoglycaemic and Streptozotocin-Induced Diabetic Rats

Energy deprivation causes neuronal death affecting the cognitive and memory ability of an individual. The kinetic parameters of glutamate dehydrogenase (GDH), the enzyme involved in the production of glutamate, was studied in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were studied to reveal the role of glutamate excitotoxicity. A single intrafemoral dose of streptozotocin was administered to induce diabetes. Hypoglycaemia was induced by appropriate doses of insulin subcutaneously in control and diabetic rats. The kinetic parameters V (max) and K (m) of GDH were studied spectrophotometrically at different substrate concentrations of alpha-ketoglutarate. Glutamate receptor binding assay was done with different concentrations of [3H] Glutamate. The GDH enzyme assay showed a significant increase (P < 0.001) in the V (max) of the enzyme in the cerebellum of hypoglycaemic and diabetic rat groups when compared to control. The V (max) of hypoglycaemic groups was significantly increased (P < 0.001) when compared to diabetic group. In the liver, the V (max) of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemia group when compared to control. The V (max) of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in V (max) (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats. The K (m) showed no significant change amongst the groups in cerebellum and liver. Scatchard analysis showed a significant increase (P < 0.001) in B (max) in the cerebellum of hypoglycaemic and diabetic rats when compared to control. The B (max) of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group. In hypoglycaemic groups, B (max) of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats. The K (d) of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats. There was a significant decrease (P < 0.05) in the K (d) of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. Our studies demonstrated the increased enzyme activity in the hypoglycaemic rats with increased production of extracellular glutamate. The present study also revealed increased binding parameters of glutamate receptors reflecting an increased receptor number with increase in the affinity. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration which has an impact on the cognitive and memory ability. This has immense clinical significance in the management of diabetes and insulin therapy.

P.4.02 Localized gray matter deficits in adolescents with first-episode psychosis

an increase in global, and no significant changes were found in the regions of interest studied. Although the opioid dependent patients craved at a high level there was no association with opioid receptor availability. However, in the alcohol dependent group, an increase in craving was significantly associated with increased availability of opioid receptors in the striatum and whole brain. We have shown an increase in opioid receptors in early abstinence from dependent alcohol and opioid use. Although the results in our alcohol dependent group did not reach significance they are consistent with another recent study (Heinz et al., 2005). This finding is most likely due to an acute increase in receptor number in early abstinence.

Regulation of 5-HT 2A/C receptors and DOI-induced behaviors by protein kinase Cγ

Protein kinase Cγ (PKCγ) null mutant mice demonstrate increased behavioral impulsivity and ethanol consumption. Pharmacological studies have shown that 5-HT 2A/C receptors modulate impulsivity and ethanol consumption in rodents and that PKC can regulate 5-HT 2A/C receptors. To determine whether PKCγ plays a selective role in 5-HT 2A/C receptor regulation, biochemical and behavioral experiments were performed in PKCγ mutant and wild-type mice. DOI-stimulated phosphoinositol hydrolysis and [ 125I]-DOI saturation binding in the PFC, and quantitative autoradiography of [ 125I]-DOI binding sites in 15 brain regions were analyzed. DOI-induced head twitch responses (HTR) were measured in naive mice after an acute 2.5 mg/kg injection of DOI. Results indicated that DOI-induced HTR was significantly greater in mutant mice compared to wild-type mice. Results of the phosphoinositol hydrolysis, membrane binding, and autoradiography experiments indicated that in mutant mice, increased HTR was associated with increased 5-HT 2A/C receptor function in the PFC, but not increased receptor number or affinity suggesting that PKCγ regulates receptor function but not receptor number. These data support a role for 5-HT 2A/C receptors in the PFC in mediating some of the behavioral differences observed between PKCγ mutant and wild-type mice.

Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors

Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between alpha2-adrenergic receptors (alpha2R) and various combinations of Gialpha and Gbeta subunit isoforms by measuring changes in FRET between Gialpha-yellow fluorescent protein and cyan fluorescent protein-Gbeta chimeras in HeLa cells. All combinations of Gialpha1, -2, or -3 with Gbeta1, -2, or -4 were activated to some degree by endogenous alpha2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Gialpha and Gbeta subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of alpha2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express alpha2a- and alpha2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the alpha2aR subtype minimized the differences among coupling preferences for Gialpha and Gbeta isoforms. The molecular properties of each Gialpha, Gbeta, and alpha2-adrenergic receptor subtype influence signaling efficiency for the alpha2-adrenergic receptor-mediated signaling pathway.

Corticosteroids: Potential 2-Agonist and Anticholinergic Interactions in Chronic Obstructive Pulmonary Disease

Corticosteroids are often used in combination with beta2-agonist and anticholinergic bronchodilators in the treatment of chronic obstructive pulmonary disease (COPD). Corticosteroids activate the beta2-receptor gene, increasing receptor number and decreasing desensitization. Long-acting beta2-agonists prime the glucocorticoid receptor and enhance nuclear translocation via activation of CCAAT enhancer binding protein-alpha. Corticosteroids can also increase prejunctional auto-inhibitory M2-receptor gene expression in airway smooth muscle. There is evidence of a synergistic inhibition of cytokine and chemokine release from alveolar macrophages, epithelial cells, and mucosal glands and enhanced respiratory cytoprotection against viral and bacterial infection when a corticosteroid is combined with salmeterol. In airway smooth muscle, corticosteroids inhibit the contractile effects of acetylcholine, whereas M2-receptor antagonism increases the relaxant activity of isoproterenol. Complementary interactions between corticosteroids and long-acting beta2-agonists and between corticosteroids and anticholinergic bronchodilators may be important if these drugs are combined in the treatment of COPD.

Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-κB in A2A adenosine receptor induction

The A(2A) adenosine receptor (A(2A)AR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A(2A) agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism initiated through NF-kappaB to terminate the activation of human and mouse macrophages.

Interaction between simvastatin and metoprolol with respect to cardiac β-adrenoceptor density, catecholamine levels and perioperative catecholamine requirements in cardiac surgery patients

Beta-blockade is a standard cardiovascular therapy known to induce the up-regulation of beta-adrenoceptor density. Upon ligand-binding, beta-adrenoceptors are normally internalised via the arrestin pathway, and after dissociation they are re-inserted into the membrane. This means that at high catecholamine levels the adrenoceptor density is low and under beta-blockade it is high. The insertion of receptors into the membrane is often dependent on farnesylation processes that can be inhibited by statins. We carried out a prospective, controlled, observational study to determine whether beta-blockade-induced up-regulation of beta-adrenoceptor density is attenuated by statin therapy and whether this would subsequently affect catecholamine consumption during surgery. We obtained pre-operative blood samples and intra-operative biopsies of the right atrial appendage from 39 patients (age: 65+/-5 years; BMI: 28+/-1) undergoing coronary bypass surgery with or without simvastatin (20 mg/day) therapy and with or without concomitant metoprolol therapy (50 mg/day). The atrial tissue was used for radioligand-binding studies with (-)-[125I]-iodocyanopindolol (ICYP) and for assessment of the beta-adrenoceptor subtype distribution following standard protocols. In the blood samples, plasma adrenaline and noradrenaline concentrations were determined using HPLC. In all tissue samples, we found a total beta-adrenoceptor density of 38+/-4 fmol/mg protein in untreated controls; this which was up-regulated to 55+/-5 fmol/mg protein in patients receiving metoprolol. This increase in receptor number was nearly prevented completely by simvastatin therapy (42+/-5 fmol/mg protein). The up-regulation could be attributed to increases in the beta1-adrenoceptor subtype. In contrast, simvastatin alone had no effect on beta-adrenoceptor density. Pre-operative adrenaline levels were slightly reduced in all drug therapy groups (nonsignificant differences), while the levels noradrenaline were not significantly different among the groups. With respect to the perioperative catecholamine requirements, patients on metoprolol needed significantly less dopamine than control patients, while patients undergoing simvastatin/metoprolol therapy needed as much as the controls. The post-operative total catecholamine requirements were not different among the four groups of patients. There were no differences in plasma metoprolol concentration between patients receiving metoprolol alone and those receiving a combination of metoprolol and simvastatin. Simvastatin therapy seems to counter-regulate the up-regulation of beta-adrenoceptor density. In the up-regulated state induced by metoprolol therapy, the patients seemed to need less catecholamines during cardiac surgery, which may be due to the higher number of beta-adrenoceptors. Additional simvastatin therapy did not reduce post-operative catecholamine consumption.

Platelet Serotonergic Markers as Endophenotypes for Obsessive-Compulsive Disorder

Although compelling evidence has shown that obsessive-compulsive disorder (OCD) has a strong genetic component, its genetic basis remains to be elucidated. Identifying biological abnormalities in nonaffected relatives is one of the strategies advocated to isolate genetic vulnerability factors in complex disorders. Since peripheral serotonergic disturbances are frequently observed in OCD patients, the aim of this study was to investigate if they could represent endophenotypes, by searching for similar abnormalities in the unaffected parents of OCD patients. We assessed whole blood serotonin (5-HT) concentration, platelet 5-HT transporter (5-HTT) and 5-HT2A receptor-binding characteristics, and platelet inositol trisphosphate (IP3) content in a sample of OCD probands (n = 48) and their unaffected parents (n = 65), and compared them with sex- and age-matched controls (n = 113). Lower whole blood 5-HT concentration, fewer platelet 5-HTT-binding sites, and higher platelet IP3 content were found in OCD probands and their unaffected parents compared to controls. Whole blood 5-HT concentration showed a strong correlation within families (p < 0.001). The only parameter that appeared to discriminate affected and unaffected subjects was 5-HT2A receptor-binding characteristics, with increased receptor number and affinity in parents and no change in OCD probands. The presence of peripheral serotonergic abnormalities in OCD patients and their unaffected parents supports a familial origin of these disturbances. These alterations may serve as endophenotypic markers in OCD, and could contribute to the study of the biological mechanisms and genetic underpinnings of the disorder.

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed alpha(4)beta(2) nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested.

Bile-pancreatic juice exclusion increases cholinergic M3 and CCK-A receptor expression and interleukin-6 production in ligation-induced acute pancreatitis

Background Using an original model, the Donor Rat Model, we showed that bile-pancreatic juice (BPJ) exclusion from gut exacerbates ligation-induced acute pancreatitis in rats. We also showed that muscarinic cholinergic M3 and CCK-A receptor expression is induced following duct ligation. Increased receptor number potentially could exacerbate cytokine production. We hypothesize that BPJ exclusion is responsible for M3 and CCK-A receptor induction and increased interleukin-6 (IL-6) production. Methods M3 and CCK-A receptor expression and IL-6 production were compared in rat pancreata 1 to 3 hours after duct ligation with or without BPJ replacement. Results Our studies showed that BPJ replacement attenuates duct ligation–induced increases in M3 and CCK-A receptor expression and IL-6 production. Conclusions In this model, BPJ exclusion from gut induces M3 and CCK-A receptor expression and increases IL-6 production. In this experimental corollary of gallstone pancreatitis, BPJ exclusion from gut may play a key role in the mechanism of disease pathogenesis.

Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na +,K +-ATPase in rats

This study demonstrated that chronic aspartame consumption in rats can lead to altered T-maze performance and increased muscarinic cholinergic receptor densities in certain brain regions. Control and treated rats were trained in a T-maze to a particular side and then periodically tested to see how well they retained the learned response. Rats that had received aspartame (250 mg/kg/day) in the drinking water for 3 or 4 months showed a significant increase in time to reach the reward in the T-maze, suggesting a possible effect on memory due to the artificial sweetener. Using [ 3H]quinuclidinyl benzilate (QNB) (1 nM) to label muscarinic cholinergic receptors and atropine (10 −6 M) to determine nonspecific binding in whole-brain preparations, aspartame-treated rats showed a 31% increase in receptor numbers when compared to controls. In aspartame-treated rats, there was a significant increase in muscarinic receptor densities in the frontal cortex, midcortex, posterior cortex, hippocampus, hypothalamus and cerebellum of 80%, 60%, 61%, 65%, 66% and 60%, respectively. The midbrain was the only area where preparations from aspartame-treated rats showed a significant increase in Na +,K +-ATPase activity. It can be concluded from these data that long-term consumption of aspartame can affect T-maze performance in rats and alter receptor densities or enzymes in brain.

IGF-1 receptor contributes to the malignant phenotype in human and canine osteosarcoma.

To further define the role of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential and three canine OS-derived cell lines were studied. Cell lines were evaluated for: IGF-1R expression; expression of IGF binding proteins (IGFBPs); effect of IGF-1 on tumor cell growth, invasion, expression of urokinase plasminogen activator (uPA), and soluble uPA receptor (suPAR), and; ectopic and orthotopic tumorigenicity of the canine OS cells in athymic mice. All cell lines exhibited steady-state mRNA expression of IGF-1R. The SAOS-2 and SAOS-LM2 cells expressed 9,138 and 10,234 cell-associated binding sites, respectively. Canine OS cells expressed from 1,728 to 3,883 binding sites. Two IGF-1-treated cell lines displayed enhanced proliferation. Two cell lines formed colonies in semisolid media, and IGF-1 increased colony number. Matrigel invasion was enhanced in one cell line following IGF-1 treatment. uPA and suPAR were unchanged in SAOS-2 and SAOS-LM2 cells following IGF-1 treatment, but the highly metastatic OS line SAOS-LM2 expressed five times more suPAR and displayed enhanced invasion compared to the parental, low metastatic SAOS-2. IGFBP-5 was detected in four of five cell lines, and IGFBP-3 was detected in two canine OS cell lines. Two canine OS lines were tumorigenic, and one metastasized spontaneously. In conclusion, OS cells express IGF-1R, which can contribute to their growth and invasion. There is suggestive evidence that increasing receptor number may contribute to in vivo tumorigenesis. Additional studies are needed to determine how IGF-1/IGF-1R interactions contribute to the malignant phenotype of OS.

Cocaine treatment causes early and long-lasting changes in muscarinic and dopaminergic receptors.

1. The study of changes that persist after drug discontinuation could be fundamental to understand the mechanisms involved in craving and relapse. 2. In this work the changes occurring in muscarinic, D1- and D2-like receptors after 30 min (immediate), 1 day (early), 5 and 30 days (late) withdrawal periods were studied, in the striatum of rats treated once a day for 7 days with cocaine 20 and 30 mg/kg, i.p. 3. Binding assays were performed in 10% homogenates and ligands used were [3H]-N-methylscopolamine, [3H]-SCH 23390, and [3H]-spiroperidol for muscarinic (M1 + M2-like), D1-, and D2-like receptors, respectively. 4. Muscarinic receptors presented a downregulation at all doses and discontinuation times, while the dissociation constant (Kd) for this receptor decreased after 30 min, 5 and 30 days abstinence times. In relation to D1-like receptors we found an antagonistic effect with 100% increase in receptor number 30 min after the last cocaine injection, but after 1-day withdrawal a downregulation was observed with both doses that persisted up to 30 days, only with the higher dose. The dissociation constant value (Kd) for this receptor showed a decrease only with 5 and 30 days withdrawal. An increase occurred with D2-like receptors at all doses and withdrawal periods studied, while Kd increased in 30-min, 5, and 30 days withdrawal. 5. In this work we found that the subchronic cocaine treatment produces early and long-lasting modifications in cholinergic muscarinic as well in dopaminergic receptors that persist up to 30 days of cocaine withdrawal.