Abdu Adem

Do tetrahydroaminoacridine (THA) and physostigmine restore acetylcholine release in Alzheimer brains via nicotinic receptors

In the presence of 9-amino-1, 2, 3,4-tetrahydroacridine (THA) 10−4M or physostigmine 10−4 M, the in vitro3H-Acetylcholine (3H-ACh) release from control cortical slices was significantly reduced. In contrast, THA 10−4 M and physostigmine 10−4 M significantly increased the release of3H-ACh in AD/SDAT brain tissue. This facilitating effect on3H-ACh release was partially blocked (50%) in the presence of the nicotinic antagonist d-tubocurarine 10−6 M indicating a possible interaction via nicotinic receptors. The muscarinic antagonist atropine 10−5 M significantly increased the3H-ACh release both in control and AD/SDAT brains, thus indicating preservation of muscarinic autoreceptors in the AD/SDAT cortical tissue. In receptor competition studies with3H-nicotine,3H-ACh and3H-quinuclidinyl benzilate (3H-QNB) as receptor ligands, THA interfered with both nicotinic and muscarinic receptor ligand binding, while physostigmine had much less effect.

Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation ☆

The specific binding of the muscarinic ligand [3H](-)quinuclidinyl benzilate [( 3H]QNB) to cell membranes of human SH-SY5Y neuroblastoma cells was studied. Saturation isotherms yielded a Kd = 0.28 +/- 0.06 nM and a Bmax of 337 +/- 47 pmol/g protein. Pirenzepine inhibited [3H]QNB binding; inhibition data showed best fit to a 2-site binding model revealing both a high affinity pirenzepine site (34%, KH = 10 nM) and a low affinity site (66%, KL = 1 microM). These results indicate that muscarinic receptors on SH-SY5Y cells may be subclassified as M1/M2 subtypes. Morphological and biochemical differentiation of these cells after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA) resulted in a decrease and an increase in the number of muscarinic binding sites, respectively. Furthermore, TPA- and RA-treated cells showed a significant increase in acetylcholinesterase activity compared with non-treated cells. However, only RA-treated cells showed significant increase in choline acetyltransferase activity compared to non-treated cells. These findings demonstrate that TPA and RA can regulate both the number of muscarinic receptors and the acetylcholinesterase activity in human SH-SY5Y neuroblastoma cells.

Change in nicotinic receptor subtypes in temporal cortex of Alzheimer brains

Competition experiments using (-)-[3H]nicotine and unlabelled nicotine revealed both high and low affinity nicotinic binding sites in temporal cortex of control and Alzheimer (AD/SDAT) brains. A significant reduction in the proportion of high affinity nicotinic binding sites (-20%) and a parallel increase in the proportion of low affinity nicotinic binding sites was obtained in AD/SDAT brain cortex compared to control brain. Moreover, a marked decrease was observed in the affinity of the low affinity nicotinic binding sites in AD/SDAT.

Extraneural cholinergic markers in Alzheimer's and Parkinson's disease

Muscarinic and nicotinic binding sites were analysed in lymphocytes from patients with Alzheimers disease, Multi-infarct dementia, Parkinsons disease and age- matched controls. A significant decrease in the number of both muscarinic and nicotinic binding sites was obtained in lymphocytes from Alzheimer patients while in Parkinson patients a significant decrease was found only in the nicotinic binding sites. Using butyrylthiocholine as substrate, no change was observed in cholinesterase activity in plasma from Alzheimer patients, whereas a significant decrease in plasma cholinesterase activity was found in Parkinson patients.

Subchronic treatment of rats with nicotine: Interconversion of nicotinic receptor subtypes in brain

A significant increase in the number of cortical high-affinity (-)-[3H]nicotine binding sites was measured in rats treated with nicotine (0.45 mg/kg) twice daily for 18 days. Competition experiments with (-)-[3H]nicotine and various concentrations of unlabelled (-)-nicotine revealed that the proportion of high-affinity nicotine binding sites was significantly increased in the nicotine-treated group while the proportion of low-affinity nicotinic binding sites was similarly significantly reduced compared to the controls. In addition there was a significant decrease in the affinity of both subtypes of nicotinic binding sites.

Distribution of nicotinic receptors in human thalamus as visualized by3H- nicotine and3H-acetylcholine receptor autoradiography

Nicotinic cholinergic receptors in human thalamus were measured using (−)3H-nicotine (20 nM) and3H-acetylcholine (3H-ACh) (20 nM) as radioligands. The specific binding for3H-nicotine to homogenates of thalamus was 51.6±8.3 pmol/g protein and for3H-ACh 18.6±1.9pmol/g protein. Receptor autoradiography indicated a high labelling of both3H-Nicotine and3H-ACh in the antero-ventral nucleus of thalamus and dorso-medial nucleus of thalamus, while the labelling was lower in the postero-lateral nucleus of thalamus and in the postero-lateral ventral nucleus of thalamus. Quantitative measurement of the3H-nicotine autoradiograms showed highest labelling in the anteroventral nucleus of thalamus (17.34±0.76pmol/g tissue). This study indicates a heterogenous distribution of high-affinity nicotinic receptors in the human thalamus.

Heterogenous Cholinergic Nicotinic Receptors in the CNS

Multiple nicotinic binding sites are found in mammalian brain. Several nicotinic agonists and antagonists have been used to characterize the nicotinic binding sites. Observations made in brain tissue homogenates and by thin tissue receptor autoradiography indicate that nicotinic antagonists and agonists might not label identical sites. A heterogeneity in agonist and antagonist binding sites is also found. Nicotinic cholinergic agonists such as 3H-nicotine and 3H-acetylcholme (3H-ACh) show a regional co-localization in mammalian brain. Nicotinic antagonists such as tubocurarine and dihydro-β-erytroidine bind to nicotinic binding sites with a regional distribution different from that of the antagonist α-bungaro-toxin. Recent studies on nicotinic receptor gene proteins and immunological studies further support the view of multiple nicotinic receptors in the CNS. Some aspects on the nicotinic receptor heterogeneity in mammalian brain will be presented in this paper.

In vitro binding of 3H-acetylcholine to nicotinic receptors in rodent and human brain.

The binding of3H-acetylcholine (3H-ACh) to nicotinic receptors in rodent and human brain was measured in the presence of atropine to prevent binding to muscarinic binding sites.3H-ACh binds specifically and saturably to rodent brain. From saturation binding Kd was 30 nM in rat cerebral cortex, which is close to that calculated from kinetic experiments. The binding was temperature-dependent, being highest at low temperatures and decreasing at higher temperatures. The regional distribution of binding in mouse brain was not uniform. The binding was highest in the midbrain, intermediate in the cerebral cortex and striatum, and lowest in the cerebellum, hippocampus, hypothalamus and medulla oblongata. No significant correlation was found between the regional3H-ACh binding and the regional binding of3H-alpha-bungarotoxin (3H-BTX),3H-nicotine (3H-NIC),3H-tubocurarine and the endogenous acetylcholine content, although the correlation value for3H-ACh/3H-NIC was at the limit for significance.3H-ACh also bound specifically to human cerebral cortical tissue and this binding was approximately three times lower than in rodent brain, when a low3H-ACh concentration was used. In contrast to rat brain there appears to exist multiple binding sites for3H-ACh in human cerebral cortex as suggested by the curvelinear nature of the Scatchard plot. It was calculated that3H-ACh bound with Kd 4 nM and Bmax 8 pmol/g protein and Kd 112 nM and Bmax 67 pmol/g protein. The Hill number of 1.5 for the binding of low concentration and 2.5 for high concentration of3H-ACh also suggest that the3H-ACh-binding sites interaction exhibit positive cooperativity.

The role of IL-12 and TNF-alpha in AIDP and AMAN.

Background:  Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) have been described as two major subtypes of Guillain‐Barré syndrome (GBS); however, the possible difference of their immune‐inflammatory pathogenesis remains unclear.

Characterization of agonist and antagonist binding to muscarinic cholinergic receptors solubilized from rat cerebral cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by the zwitter-ionic detergent, 3-((3-cholamidopropyl)dimethylamino)1-propane sulfonate (CHAPS). The supernatant, after centrifugation at 100,000 × g, was shown to contain molecules with binding sites for both3H-pirenzepine (3H-PZ) and3H-(-) quinuclidinyl benzilate (3H-QNB). Maximum binding values for3H-PZ and3H-QNB binding to solubilized receptors were approximately 176±24 pmol/g and 370±53 pmol/g of protein, respectively. The Kd values for3H-PZ and3H-QNB binding to solubilized receptors were 27±6.3nM and 0.17±0.03 nM, respectively. The rank order of potencies of muscarinic drugs, in terms of their ability to inhibit binding of either3H-PZ or3H-QNB, was atropine > pirenzepine > oxotremorine > carabachol. Pirenzepine inhibited3H-QNB binding with a Hill coefficient of 0.77, but inhibited3H-PZ with a Hill coefficient of 0.94. Hill coefficients for agonists were less than 1. These findings indicate that muscarinic receptors solubilized from rat brain cortex retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.