Variations in binding affinities of the peripheral nicotinic acetylcholine receptor can be attributed to endogenous acetylcholine

Equilibrium binding studies performed with fresh membrane fragments from Torpedo marmorata reveal a low affinity for [3H]acetylcholine with an equilibrium dissociation constant in the micromolar range and no indication of cooperative interactions. The low binding affinity is an artifact caused by the presence of endogenous acetylcholine and is not related to the active conformation of the receptor. Endogenous acetylcholine is identified by its interaction with acetylcholine esterase and choline kinase. It is present in presynaptic vesicles as shown in electron micrographs. Leakage of these synaptosomes is of the order of 300 pmol acetylcholine per g tissue as determined by means of binding studies performed with [3H]acetylcholine. In the absence of endogenous acetylcholine, equilibrium binding studies show a high affinity for [3H]acetylcholine and a slight cooperativity of sites (K1D = 30nM; K2D = 10nM). The addition of detergents, local anesthetics or alcohols to a further increase in affinity and to a decrease in cooperativity (K1D = 11nM; K2D = 5nM). No low-affinity binding can be detected in the micromolar range.     

Variations on fibrinogen-erythrocyte interactions during cell aging

Erythrocyte hyperaggregation, a cardiovascular risk factor, is considered to be caused by an increase in plasma adhesion proteins, particularly fibrinogen. We have recently reported a specific binding between fibrinogen and an erythrocyte integrin receptor with a β(3) or β(3)-like subunit. In this study we evaluate the influence of erythrocyte aging on the fibrinogen binding. By atomic force microscopy-based force spectroscopy measurements we found that increasing erythrocyte age, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence but not its force. This observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. We conclude that upon erythrocyte aging the number of fibrinogen molecules bound to each cell decreases significantly, due to the progressive impairment of the specific fibrinogen-erythrocyte receptor interaction. Knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen content in blood, which could disturb the blood flow. Our data also show that the sialic acids exposed on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating its binding to the erythrocyte membrane receptor.     

Glucose catabolism during aging and differentiation in hypocotyls ofPhaseolus mungo seedlings

Changes in activities of the glycolytic and pentose phosphate (PP) pathways in glucose catabolism in various parts of the hypocotyls obtained from 4-day-old etiolatedPhaseolus mungo seedlings were investigated by measuring the inhibition rates of respiration by iodoacetate and malonate, and the release of¹⁴CO₂ from [1-¹⁴C]- and [6-¹⁴C]glucose. The relative activity of the PP pathway in glucose catabolism was higher in the immature part (Part I) and the aged part (Part V) of the hypocotyls than in the intermediary one (Part III), while the activity of the glycolytic pathway decreased with aging. On a fresh weight basis, the enzyme activities of the glycolytic and PP pathways were higher in Part I than in Parts III and V. On a protein content basis, however, activities of the enzymes of the PP pathway increased with aging and differentiation of the hypocotyls whereas those of the glycolytic pathway decreased. Levels of nicotinamide adenine nucleotides were found to be in the following order: Part I>Part III> Part V for NAD⁺+NADH; Part I>Part V>Part III for NADP⁺+NADPH. The stimulative effect of methylene blue on decreasing the C₆/C₁ ratio was greater in Part III than in Part I, and No effect was observed in Part V. These data suggest that a decrease in the activity of the glycolytic pathway with aging and differentiation may be due to the decreasing glycolytic enzyme activities and NAD(H) content. The higher activity of the PP pathway in the immature part is attributable to larger amounts of NADP(H) and enzymes of the PP pathway. The greater contribution of the PP pathway to glucose catabolism in the aged part than in the intermediary part seems to results from a more active turnover of NADP and the relatively higher activity of the enzymes of the PP pathway than those of the glycolytic pathway.     

Muscarinic acetylcholine receptor in chick limb bud during morphogenesis

Summary In the chick embryo a cholinesterase activity appears in various organ anlagen which has been correlated with morphogenetic movements (Drews 1975). The cholinesterase activity is present in the mesenchyme of the limb bud during aggregation of the central chondrogenic core. In the present study binding of tritium labelled quinuclidinyl benzilate ((³H)QNB), a muscarinic antagonist, to homogenates of chick limb buds was investigated by a filtration assay. In the homogenate of limb buds at Stage 24 specific binding of (³H)QNB was demonstrated. Determination of binding constants and inhibition of binding by agonists and antagonists was studied at Stage 25/26. Specific binding was defined by the difference in binding in the absence and presence of atropine (1 M). Specific binding of (³H)QNB reflected a muscarinic receptor. The K_d in two experiments was 0.11 nM and 0.16 nM, the binding capacity was 15.7 fmol (³H)QNB/mg protein and 12.0 fmol (³H)QNB/mg protein, respectively. Data on displacement of specific bound (³H)QNB by various nicotinic and muscarinic ligands confirmed the muscarinic nature of the receptor. Muscarinic ligands inhibited the (³H) QNB binding, whereas nicotinic ligands caused no inhibition at pharmacological concentrations. I conclude that a specific muscarinic acetylcholine receptor is part of the cholinergic system whose presence is indicated by cholinesterase activity in the chondrogenic core of the limb bud during morphogenesis.     

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating

The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closed<==>open isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (phi) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. phi was constant throughout the length of the alpha subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two alpha subunits move synchronously. Between subunits, the sequence of M4 motions is alpha-epsilon-beta. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.     

Inflammatory cytokines decrease the expression of nicotinic acetylcholine receptor during the cell maturation

It is known that the nervous system significantly attenuates systemic inflammatory responses through the parasympathetic nervous system. Furthermore, it has been reported that the alpha7 subunit of a nicotinic acetylcholine receptor is required for a cholinergic inhibition against cytokine synthesis in a macrophage. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses and the maintenance of immunity, in this study, we investigated the expression level of nicotinic receptors of a p53-deficient APC cell line (JawsII) derived from a mouse bone marrow. We showed that stimulation of the JawsII cells with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) led increase of CD80 and CD86 expression while diminishment of the surface nicotinic receptor. On the other hand, stimulation of nicotinic receptor had no effect on these phenomena. Furthermore, we examined the ability of the cells to release cytokine when stimulated with both nicotine and LPS and showed that the stimulation with LPS augmented the secretion of IL-1a, IL-1b, IL-6, and TNF-α. These results suggested that nicotinic stimulation had no effect on the diminishment of alpha7 nicotinic acetylcholine receptor on JawsII cells by LPS stimulation.     

Variations in ATP synthetase activity of rat liver mitochondria during aging and under the effect of thyroxine

Experiments with 1-, 3-, 12- and 24-month male rats show a decrease in the rate of ATP synthesis by the liver mitochondria with oxidation of succinate and alpha-glycerophosphate. Administration of thyroxin to animals for 2 and 9 days evokes a more pronounced increase of ATP-synthetase activity in 12- and 24-month rats and an essential decrease of the age differences of the activity. It is suggested that the character of observed variations is caused by age changes in the system of the hormonal metabolism control.     

Regulation of acetylcholine receptor channel function during development of skeletal muscle

The nicotinic acetylcholine (ACh) receptor channel mediates synaptic transmission at the neuromuscular junction. During the development of skeletal muscle, ACh receptors undergo changes in distribution, antigenic determinants, degradation rate, and function. Now that these developmental hallmarks have been identified, attention has turned toward understanding both the structural bases for such changes and the role of nerve in triggering these changes. Recently, a much clearer understanding of one of these developmental processes, namely, the alterations in channel function, has emerged through both sensitive patch-clamp measurements and the application of recombinant DNA technology. In light of these new advances, we now reevaluate the processes governing the developmental changes in the functional properties of the ACh receptor.     

Phosphorylation of phosphatidylinositides during muscarinic acetylcholine receptor regulation of myocardium contraction

While estimating the myocardium sarcolemma phosphatidylinositides phosphorylation the muscarime acetylcholine receptor agonist carbacholine (10(-7) M) was determined as capable to stimulate 32P incorporation into phosphatidylinositol-4-monophosphate (in 2.6 times) and phosphatidylinositol-4,5-bisphosphate (in 2.3 times) indicating to the activation of phosphatidylinosite kinase and phosphatidylinosite 4-kinase respectively. The phosphorylation reactions in general completely depend on the presence in the incubation medium of Mg2+ capable in 10 mkM concentration to increase 32P influx into phosphatidylinositol-4-monophosphate 8 times, and into phosphatidylinositol-4,5-bisphosphate 4 times. Carbacholine (10(-7) M) also activates phospholipase C which hydrolyses phosphatidylinositol-4,5-bisphosphate. The latter is substantiated by the increase (2.6 times) of the secondary messenger--inositol-1,4,5-trisphosphate formation.     

Conformational dynamics of the alphaM3 transmembrane helix during acetylcholine receptor channel gating

Muscle acetylcholine receptors are synaptic ion channels that "gate" between closed- and open-channel conformations. We used Phi-value analysis to probe the transition state of the diliganded gating reaction with regard to residues in the M3, membrane-spanning helix of the muscle acetylcholine receptor alpha-subunit. Phi (a fraction between 1 and 0) parameterizes the extent to which a mutation changes the opening versus the closing rate constant and, for a linear reaction mechanism, the higher the Phi-value, the "earlier" the gating motion. In the upper half of alphaM3 the gating motions of all five tested residues were temporally correlated (Phi approximately 0.30) and serve to link structural changes occurring at the middle of the M2, pore-lining helix with those occurring at the interface of the extracellular and transmembrane domains. alphaM3 belongs to a complex and diverse set of synchronously moving parts that change structure relatively late in the channel-opening process. The propagation of the gating Brownian conformational cascade has a complex spatial distribution in the transmembrane domain.     

Insulin receptor kinase activity in rat adipocytes is decreased during aging

The tyrosine kinase activity of the insulin receptor derived from rat adipocyte plasma membranes was examined during aging. In the absence of insulin, autophosphorylation and histone H2B phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among 3- and 24-month-old rats. In contrast, insulin-stimulated kinase activity was significantly reduced in the old animals. We have also found that the insulin dependent phosphorylation of a putative endogenous substrate of 60 kDa was drastically reduced in old animals. These results suggest that the decrease in kinase activity in old rats could be related with the insulin resistance of aging.