Presynaptic alpha7-nicotinic acetylcholine receptors mediate nicotine-induced nitric oxidergic neurogenic vasodilation in porcine basilar arteries.

We previously reported that nicotine-induced nitric oxide (NO)-mediated neurogenic vasodilation in the porcine basilar artery was dependent on intact sympathetic innervation. We further demonstrated in this artery that nicotine acted on nicotinic acetylcholine receptors (nAChRs) on presynaptic sympathetic nerve terminals to release norepinephrine (NE), which then acted on beta2-adrenoceptors located on the neighboring NOergic nerve terminals to release NO, resulting in vasodilation. The nature of the nAChRs has not been determined. The nAChR subtype mediating nicotine-induced dilation in isolated porcine basilar arterial rings denuded of endothelium was therefore examined pharmacologically and immunohistochemically. Results from using an in vitro tissue bath technique indicated that relaxation induced by nicotine (100 microM) was blocked by preferential alpha7-nAChR antagonists (methyllycaconitine and alpha-bungarotoxin) and nonspecific nAChR antagonist (mecamylamine) in a concentration-dependent manner, but was not affected by dihydro-beta-erythroidine (a preferential alpha4-nAChR antagonist). These nAChR antagonists did not affect relaxation elicited by transmural nerve stimulation (8 Hz) or that by sodium nitroprusside and NE. Results from double-labeling immunohistochemical studies in whole-mount porcine basilar and middle cerebral arteries and in cultured porcine superior cervical ganglia (SCG) indicated that alpha7-nAChR- and tyrosine hydroxylase immunoreactivities were colocalized in same nerve fibers. These results suggest the presence of functional alpha7-nAChRs on postganglionic sympathetic adrenergic nerve terminals of SCG origin, which mediate nicotine-induced neurogenic NOergic vasodilation. These findings are consistent with our hypothesis that nicotine acts on nAChRs on presynaptic sympathetic nerve terminals to release NE, which then acts on presynaptic beta2-adrenoceptors located on the neighboring NOergic nerve terminals, resulting in release of NO and dilation of porcine basilar arteries.     

Differential effects of endogenous and synthetic cannabinoids on alpha7-nicotinic acetylcholine receptor-mediated responses in Xenopus Oocytes

The effects of endogenous and synthetic cannabinoid receptor agonists, including 2-arachidonoylglycerol (2-AG), R-methanandamide, WIN55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], and CP 55,940 [1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol], and the psychoactive constituent of marijuana, Delta9-tetrahydrocannabinol (Delta9-THC), on the function of homomeric alpha7-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. The endogenous cannabinoid receptor ligands 2-AG and the metabolically stable analog of anandamide (arachidonylethanolamide), R-methanandamide, reversibly inhibited currents evoked with ACh (100 microM) in a concentration-dependent manner (IC50 values of 168 and 183 nM, respectively). In contrast, the synthetic cannabinoid receptor agonists CP 55,940, WIN55,212-2, and the phytochemical Delta9-THC did not alter alpha7-nACh receptor function. The inhibition of alpha7-mediated currents by 2-AG was found to be non-competitive and voltage-independent. Additional experiments using endocannabinoid metabolites suggested that arachidonic acid, but not ethanolamine or glycerol, could also inhibit the alpha7-nACh receptor function. Whereas the effects of arachidonic acid were also noncompetitive and voltage-independent, its potency was much lower than 2-AG and anandamide. Results of studies with chimeric alpha7-nACh-5-hydroxytryptamine (5-HT)3 receptors comprised of the amino-terminal domain of the alpha7-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT3 receptors indicated that the site of interaction of the endocannabinoids with the alpha7-nAChR was not located on the N-terminal region of the receptor. These data indicate that cannabinoid receptor ligands that are produced in situ potently inhibit alpha7-nACh receptor function, whereas the synthetic cannabinoid ligands, and Delta9-THC, are without effect, or are relatively ineffective at inhibiting these receptors.     

Additive effects of endogenous cannabinoid anandamide and ethanol on alpha7-nicotinic acetylcholine receptor-mediated responses in Xenopus Oocytes

The interaction between the effects of the endogenous cannabinoid receptor agonist anandamide and ethanol on the function of homomeric alpha(7)-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes were investigated using the two-electrode voltage-clamp technique. Anandamide and ethanol reversibly inhibited currents evoked with 100 microM acetylcholine in a concentration-dependent manner. Coapplication of anandamide and ethanol caused a significantly greater inhibition of alpha(7)-nACh receptor function than anandamide or ethanol alone. The IC(50) value of 238 +/- 34 nM for anandamide inhibition decreased significantly to 104 +/- 23 nM in the presence of 30 mM ethanol. The inhibition of alpha(7)-mediated currents by coapplication of anandamide and ethanol was not altered by phenylmethylsulfonyl fluoride, an inhibitor of anandamide hydrolyzing enzyme, or N-(4-hydroxyphenyl)-arachidonylamide, an anandamide transport inhibitor. Analysis of oocytes by matrix-assisted laser desorption/ionization technique indicated that ethanol treatment did not alter the lipid profile of oocytes, and there is negligible, if any, anandamide present in these cells. Results of studies with chimeric alpha(7)-nACh-5-HT(3) receptors comprised of the amino-terminal domain of the alpha(7)-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT(3) receptors suggest that although ethanol inhibition of the alpha(7)-nACh receptor is likely to involve the N-terminal region of the receptor, the site of action for anandamide is located in the transmembrane and carboxyl-terminal domains of the receptors. These data indicate that endocannabinoids and ethanol potentiate each other's inhibitory effects on alpha(7)-nACh receptor function through distinct regions of the receptor.     

Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis

OBJECTIVE: Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. DESIGN: Randomized and controlled in vitro and in vivo study. SETTINGS: Research laboratory and animal facility rooms. SUBJECTS: RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). INTERVENTIONS: RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. MEASUREMENTS AND MAIN RESULTS: GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). CONCLUSION: These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.     

The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes

The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and noncompetitive manner.     

Presynaptic muscarinic M(2)-receptor-mediated inhibition of N-type Ca(2+) channels in cultured sphenopalatine ganglion: direct evidence for acetylcholine inhibition of cerebral nitrergic neurogenic vasodilation

Results of previous pharmacological studies suggested that presynaptic muscarinic M(2) receptors on cerebral perivascular nitric oxidergic (nitrergic) nerves mediated inhibition of nitric oxide release from these nerves. The inhibition was thought to be primarily attributable to a decreased Ca(2+) influx through N-type Ca(2+) channels on nitrergic nerves, but direct evidence supporting this hypothesis was not presented. In the present study, we used cultured rat sphenopalatine ganglion (SPG), a major source of nitrergic nerves to cerebral blood vessels, to investigate the role of muscarinic M(2) receptors in modulating voltage-dependent Ca(2+) channels. SPG neuronal soma and dendrites were immunoreactive for both N-type Ca(2+) channels and muscarinic M(2) receptors, indicating that muscarinic M(2) receptors were colocalized with N-type Ca(2+) channels. Using the whole-cell voltage-clamp technique, we found that voltage-dependent Ca(2+) currents in cultured SPG were largely blocked by omega-conotoxin, an N-type calcium channel antagonist, but were not affected by nifedipine, an L-type calcium antagonist. The Ca(2+) current was inhibited by acetylcholine (ACh) and arecaidine but-2-ynyl ester tosylate (ABET), a preferential muscarinic M(2)-receptor agonist, in a concentration-dependent manner. The inhibition was reversed by atropine and methoctramine (a muscarinic M(2)-receptor antagonist), but was not affected by muscarinic M(1)-, M(3)-, or M(4)-receptor antagonists. Consistent with this, preferential muscarinic M(1)-receptor agonists McN-A-343 and oxotremorine did not affect the Ca(2+) current. Furthermore, pretreatment with pertussis toxin and guanosine 5'-O-(3-thio)triphosphate prevented ACh and ABET inhibition of Ca(2+) currents. These results are consistent with pharmacological findings in the pig basilar arteries and provide direct evidence supporting our hypothesis that M(2)-receptor-mediated inhibition of cerebral nitrergic neurogenic vasodilation is due to a G(i)-protein-mediated suppression of Ca(2+) influx via voltage-dependent N-type Ca(2+) channels on perivascular nerves.     

High-affinity epibatidine binding of functional, human alpha7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells

Human nicotinic acetylcholine receptor (nAChR) alpha7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows alpha7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled alpha-bungarotoxin (I-Bgt) binding sites corresponding to human alpha7 (halpha7)-nAChRs on the surface of most cells. I-Bgt binds to halpha7-nAChRs in membrane fractions with a typical apparent K(D) value of approximately 5 nM and B(max) value of approximately 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed halpha7-nAChRs is evident as rapid, transient inward current responses to (-)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to approximately 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing alpha3 or alpha4 subunits (K(D) values of about 100 or 10 pM, respectively). halpha7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (K(D) value of approximately 0.6 nM). Studies of several forms of native or heterologously expressed rat or human alpha7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and alpha-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine approximately cytisine > carbamylcholine approximately D-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human alpha7-nAChRs that exhibit ligand binding and functional properties like native alpha7-nAChRs and that epibatdine is useful as a probe for human alpha7-nAChRs.     

The P2X7 receptor-pannexin-1 complex decreases muscarinic acetylcholine receptor-mediated seizure susceptibility in mice

Pannexin-1 (Panx1) plays a role in the release of ATP and glutamate in neurons and astrocytes. Panx1 can be opened at the resting membrane potential by extracellular ATP via the P2X7 receptor (P2X7R). Panx1 opening has been shown to induce neuronal death and aberrant firing, but its role in neuronal activity has not been established. Here, we report the role of the P2X7R-Panx1 complex in regulating muscarinic acetylcholine 1 (M1) receptor function. P2X7R knockout (P2X7-/-) mice showed greater susceptibility to seizures induced by pilocarpine (PILO), an M1 receptor agonist, than their WT littermates, despite having similar levels of hippocampal M1 receptor expression. This hypersensitivity to PILO in the P2X7-/- mice did not involve the GABA or glutamate system. Both administration of P2X7R antagonists and gene silencing of P2X7R or Panx1 in WT mice increased PILO-induced seizure susceptibility in a process mediated by PKC via intracellular Ca2+ release. Therefore, we suggest that the P2X7R-Panx1 complex may play an important role as a negative modulator of M1 receptor-mediated seizure activity in vivo.     

Effects of prolonged nicotinic ligand exposure on function of heterologously expressed,human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptors

Effects of prolonged nicotinic ligand exposure on the function of human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EP1 human epithelial cells. Magnitudes of acute, nAChR-mediated, specific 86Rb+ efflux responses to 1 mM carbamylcholine were reduced after pretreatment with specific nAChR ligands in effects that depended on pretreatment drug dose, duration of drug pretreatment, and duration of drug-free recovery. Fifty percent inhibition of alpha4beta2-nAChR function following 5 min of recovery occurred after 1 min of pretreatment with 1 mM nicotine but also after 1-h pretreatment at 10 nM nicotine. Seventy-five percent loss in function persisted 1 h after drug removal following 15 min or more of exposure to 1 mM nicotine. However, functional recovery was nearly complete after 1 h in drug-free medium following 1 min to 24 h pretreatment with 0.1 to 1 microM nicotine, i.e., in the range of smoker plasma nicotine levels. alpha4beta4-nAChR was similarly sensitive to persistent inactivation by prolonged nicotine exposure. Carbamylcholine exhibited slightly lower persistent inactivation potency than nicotine at both alpha4beta2- and alpha4beta4-nAChR. The nAChR antagonist, mecamylamine, exhibited persistent inactivation potency and efficacy similar to nicotine at alpha4beta2-nAChR but had a reduced effect on alpha4beta4-nAChR. These studies illustrate persistent inactivation of human alpha4beta2- or alpha4beta4-nAChR induced by prolonged exposure to nicotine and show that other ligands induce nAChR persistent inactivation in a subtype-specific manner.     

Dissociation between muscarinic receptor-mediated inhibition of adenylate cyclase and autoreceptor inhibition of [3H] acetylcholine release in rat hippocampus

Activation of muscarinic cholinergic receptors (mAChRs) in the central nervous system reduces the catalytic activity of membrane-bound adenylate cyclase and attenuates depolarization-dependent release of acetylcholine (ACh). Inasmuch as reports have indicated that these mAChR-mediated responses exhibit pharmacological profiles similar to the M2 subclass of mAChR, the present studies were undertaken to ascertain whether attenuation of presynaptic adenylate cyclase activity [and concurrent reduction of intraneuronal cyclic AMP (cAMP) levels] underlies mAChR-mediated autoinhibition of electrically evoked ACh release. In [3H]choline-prelabeled rat hippocampal slices, the mAChR agonists oxotremorine (EC50 = 15 microM) and carbachol (EC50 = 80 microM) caused atropine-reversible inhibition of [3H]ACh release up to a maximum of 80% reduction. The rank order of potency for antagonist reversal of this inhibitory action (N-methylatropine = atropine greater than scopolamine much greater than pirenzepine) was generally consistent with an M2 mAChR-mediated response although pirenzepine was ineffective up to 1 mM. Under these assay conditions, forskolin (1-10 microM) and 8-bromo-cAMP (30-300 microM) enhanced electrically evoked [3H]ACh release maximally by 50 to 60%; however, neither agent significantly reversed mAChR agonist-induced inhibition of [3H]ACh release. Additional studies were undertaken to determine the consequences of chemically uncoupling mAChR from their G protein-adenylate cyclase effector system in this tissue. Whereas brief pretreatment with the sulfhydryl alkylating agent N-ethylmaleimide (30 microM) or pertussis toxin (1 microgram/ml) markedly attenuated carbachol inhibition of adenylate cyclase activity in hippocampal tissue, there was no concurrent reduction of carbachol-inhibited [3H] ACh release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of neointimal hyperplasia in porcine coronary arteries by 2-deoxy-d-glucose

Background

The drug eluting stents have been shown to play a substantial role in preventing in-stent restenosis. This study was initiated to determine the efficacy of 2-deoxy-d-glucose (2-DG) in an in-stent restenosis model for reducing neointimal hyperplasia after coronary stent placement.

Methods

In a porcine overstretch model, three kinds of stents were investigated (n = 12 per group): bare metal stents (BMS), rapamycin-eluted stents (RES), and BMS after intracoronary short-term application of 2-DG (DGS). After 42 days histomorphometric and histopathological analyses were performed.

Results

Neointimal thickness (BMS: 0.38 ± 0.08, RES: 0.24 ± 0.11, DGS: 0.15 ± 0.01), area stenosis (BMS: 47.39 ± 2.76, RES: 32.2 ± 2.08, DGS: 29.30 ± 2.98) did not differ after 42 days between the RES and DGS but were significantly lower as compared to BMS only. Lumen area (BMS: 3.15 ± 1.53, RES: 4.37 ± 1.72, DGS: 4.77 ± 2.14) was significantly higher in the DGS group in comparison to the BMS group. The calculated injury and inflammation scores were similar and re-endothelialization was confirmed in all groups.

Conclusions

This study could demonstrate that in porcine stent model neointimal hyperplasia and re-endothelialization after application of 2-DG are comparable to those seen in RES. Thus, 2-DG might be a promising clinical application for coronary stent coating.     

An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo

Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.     

Involvement of alpha 7 nicotinic acetylcholine receptors in gene expression of dopamine biosynthetic enzymes in rat brain

Brain dopaminergic systems are critical in mediating the physiological responses to nicotine. The effects of several concentrations of nicotine (0.08, 0.17, or 0.33 mg/kg body weight) and involvement of alpha7 nicotinic acetylcholine receptors (nAChRs) in gene expression of key enzymes in dopamine biosynthesis were evaluated in the ventral tegmental area (VTA) and substantia nigra (SN), cell bodies of the mesocorticolimbic and nigrostriatal pathways. Nicotine elicited a dose-dependent elevation of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis in VTA and SN. The VTA was more sensitive to lower concentrations of nicotine with maximal response observed with the lowest dose of nicotine. Nicotine also elevated mRNA levels of GTP cyclohydrolase I (GTPCH), rate limiting in biosynthesis of TH's essential cofactor tetrahydrobiopterin in both dopaminergic locations. The changes in TH and GTPCH mRNAs were correlated. Pretreatment with the alpha7 nAChR antagonist methyllycaconitine prevented the nicotine-induced rise in TH or GTPCH mRNA in VTA and SN. Administration of alpha7 nAChR agonist 3-[2,4-dimethoxybenzilidene]anabaseine at 1 to 10 mg/kg or (E,E-3-(cinnamylidene)anabaseine at 0.3 to 1 mg/kg increased TH mRNA in VTA and SN, but not in peripheral catecholaminergic cells. Thus, agonists of alpha7 nAChRs have therapeutic potential for increasing TH gene expression in dopaminergic regions without some of nicotine's disadvantages, such as its harmful effects on the cardiovascular system. The findings indicate that nicotine may regulate dopamine biosynthesis by alterations in gene expression of TH and its cofactor. The alpha7 nAChRs are involved in mediating these effects of nicotine.     

The modulating role of nuclear factor-kappaB in the action of alpha7-nicotinic acetylcholine receptor and cross-talk between 5-lipoxygenase and cyclooxygenase-2 in colon cancer growth induced by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone

4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the tobacco-specific nitrosamine, induces lung cancer in all animal species tested and is thought to contribute significantly to the high lung cancer burden associated with smoking. However, there is no report whether NNK could promote colon cancer growth. To address this hypothesis and the possible signaling pathways involved, we used SW1116 colon cancer cell line to study these biological events in vitro. Results showed that NNK, after 5-h treatment, stimulated cell proliferation, enhanced alpha7-nicotinic acetylcholine receptor (alpha7-nAChR) mRNA levels and nuclear factor-kappaB (NF-kappaB) DNA binding activity, as well as 5-lipoxygenase and cyclooxygenase-2 protein expressions. alpha-Bungarotoxin, the specific alpha7-nAChR antagonist, inhibited these biological effects. However, 5-lipoxygenase inhibition had no effect on alpha7-nAChR mRNA expression, but significantly inhibited cell proliferation and activation of NF-kappaB and cyclooxygenase-2, whereas NF-kappaB-specific inhibitor caffeic acid phenethyl ester reduced both cell proliferation and cyclooxygenase expression induced by NNK without affecting alpha7-nAChR mRNA level and 5-lipoxygenase expression. Together, the present study demonstrated that NNK promoted colon cancer growth in vitro. NF-kappaB not only conveys the biological effect of alpha7-nAChR activation but is also involved in the cross-talk between 5-lipoxygenase and cyclooxygenase-2 in response to NNK in colon cancer cell development.     

O2-sensitivity of beta adrenergic responsiveness in isolated bovine and porcine coronary arteries

The relation between pO2 and beta adrenergic responsiveness was studied in isolated bovine and porcine coronary artery rings. Isoproterenol elicited a concentration-dependent relaxation of bovine and porcine coronary artery rings precontracted with KCI (2 X 10(-2) M) or histamine (10(-6) M); beta adrenergic responsiveness was significantly lower in K+-depolarized coronary arteries. A decrease of bath pO2 from 95 to 40% significantly reduced beta adrenergic responsiveness in both coronary preparations precontracted with either KCI or histamine. Similarly, exogenous arachidonic acid (3 X 10(-6) to 3 X 10(-5) M) depressed isoproterenol-induced relaxations in both tissues. Indomethacin (5 X 10(-6) M) augmented beta adrenergic responsiveness in the presence of 95% O2 and prevented the inhibitory effects of the decrease in bath pO2 and arachidonic acid in both preparations. The experimental data suggest that the demonstrated O2-sensitivity of beta adrenergic responsiveness is mediated by vascular prostaglandin synthesis in isolated large coronary arteries.     

Methanandamide allosterically inhibits in vivo the function of peripheral nicotinic acetylcholine receptors containing the alpha 7-subunit

Methanandamide (MAEA), the stable analog of the endocannabinoid anandamide, has been proven in Xenopus oocytes to allosterically inhibit the function of the alpha7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 micromol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 micromol/kg) inhibited the electrically induced tachycardia (maximally by 15-20%; abolished by the CB(1) receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 micromol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB(1) receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 micromol/kg) and the selective alpha7-subunit antagonist methyllycaconitine (MLA; 3 and 10 micromol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the alpha7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 micromol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely alpha7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.     

Stimulation of alpha7 nicotinic acetylcholine receptor inhibits CD14 and the toll-like receptor 4 expression in human monocytes

The lipopolysaccharide (LPS)-receptor complex, CD14/toll-like receptor 4, is known to play a role in the immune responses during sepsis. Excessive inflammation and tumor necrosis factor (TNF)-alpha synthesis have been reported to cause morbidity and mortality in endotoxemia and sepsis. Cell-to-cell interaction through the engagement between intercellular adhesion molecule 1, B7.1, and CD40 on monocytes and their ligands on T cells has been suggested to play a role in the inflammatory response such as TNF-alpha and interleukin 10 production. Nicotine, with the stimulation of the nicotinic acetylcholine receptor alpha7 subunit (alpha7-nAChR), has now become the focus of attention because of its anti-inflammatory effects. However, little is known about the mechanism of the inhibitory effects induced by nicotine on the LPS-induced immune responses. In the present study, we found that nicotine suppressed the expression of CD14, toll-like receptor 4, intercellular adhesion molecule 1, B7.1, and CD40 on monocytes and the production of TNF-alpha, but not interleukin 10, in human peripheral blood mononuclear cells in the presence of LPS. The actions of nicotine were reversed by a nonselective and a selective alpha7-nAChR antagonist, mecamylamine and alpha-bungarotoxin, respectively. Therefore, nicotine might inhibit the LPS receptor complex expression via alpha7-nAChR, thus leading to a decrease in the adhesion molecule expression and TNF-alpha production. Moreover, we demonstrated that a nuclear factor-kappaB and a p38 mitogen-activated protein kinase inhibitor mimicked the actions of nicotine in the presence of LPS. These results suggested that the nuclear factor-kappaB and p38 mitogen-activated protein kinase might be involved in the actions of nicotine.