Susana Nieto-Cerón

Glucagon increases contractility in ventricle but not in atrium of the rat heart

This study evaluates the inotropic responses to glucagon in electrically driven isolated left and right atria as well as in right ventricular strips of rat heart. For comparison, the contractile effects resulting from stimulating beta-adrenoceptors with isoprenaline in atrial and ventricular tissues were also obtained. Glucagon (0.01-1 microM) produces a concentration-dependent positive inotropic effect in ventricular but not in atrial myocardium. Isoprenaline, however, increases contractility both in atrial and ventricular tissues. The nonselective phosphodiesterase (PDE) inhibitor 3-isobutylmethylxantine (IBMX, 10 microM) enhances the contractile effect of glucagon on ventricular myocardium. However, glucagon still failed to increase contractility in atrial myocardium in the presence of 10 microM, IBMX. Also, in left atria of rats pretreated with pertussis toxin, glucagon did not produce any positive inotropic effect, either alone or in the presence of 10 microM, IBMX. Western blotting analysis indicates that glucagon receptors expression is 5 times higher in ventricular than in atrial myocardium. Taken together, these results indicate that the lack of inotropic effect of glucagon in atrium is not due to Gi protein or PDEs activity but seems to be a consequence of a lower glucagon receptor density in this tissue.

Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice

Half of congenital muscular dystrophy cases arise from laminin α2 (merosin) deficiency, and merosin‐deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean ± SD 1.42 ± 0.28 µmol acetylthiocholine/h/mg protein, U/mg) was decreased by ∼50% in dystrophic thymus (0.77 ± 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT–PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (, 64%) and monomers (, 19%), as well as hydrophilic tetramers (, 9%) and monomers (, 8%). The dimers consisted of glycosylphosphatidylinositol‐anchored H subunits. Western blot assays with anti‐AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol‐specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.

Cancer-associated differences in acetylcholinesterase activity in bronchial aspirates from patients with lung cancer.

In non-neuronal contexts, ACh (acetylcholine) is thought to be involved in the regulation of vital cell functions, such as proliferation, differentiation, apoptosis and cell-cell interaction. In airways, most cells express the non-neuronal cholinergic system, each containing a specific set of components required for synthesis, signal transduction and ACh hydrolysis. The aim of the present study was determine the expression of cholinergic system components in bronchial aspirates from control subjects and patients with lung cancer. We conducted an analysis of cholinergic components in the stored soluble and cellular fraction of bronchial aspirates from non-cancerous patients and patients diagnosed with lung cancer. The results show that the fluid secreted by human lung cells contains enough AChE (acetylcholinesterase) activity to control ACh levels. Thus these findings demonstrate that: (i) AChE activity is significantly lower in aspirates from squamous cell carcinomas; (ii) the molecular distribution of AChE in both bronchial cells and fluids consisted of amphiphilic monomers and dimers; and (iii) choline acetyltransferase, nicotinic receptors and cholinesterases are expressed in cultured human lung cells, as demonstrated by RT-PCR (reverse transcriptase-PCR). It appears that the non-neuronal cholinergic system is involved in lung physiology and lung cancer. The physiological consequences of the presence of non-neuronal ACh will depend on the particular cholinergic signalling network in each cell type. Clarifying the pathophysiological actions of ACh remains an essential task and warrants further investigation.

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.

Analysis of cholinesterases in human prostate and sperm: implications in cancer and fertility.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are postulated to play non-cholinergic roles in cellular physiology. The probable implication of cholinesterases (ChEs) in several human pathologies prompted us to study the cholinergic components in the male reproductive system. Surgical pieces of prostatic cancer (PC) and benign prostatic hyperplasia (BPH) were analyzed for AChE and BChE activity. Loosely (S1) and tightly (S2) bound AChE and BChE forms were characterized by sedimentation analysis. The mean AChE activity in BHP samples was 2.38+/-0.56 mU/mg (nmol of the substrate hydrolysed per minute and per milligram protein) and 2.57+/-0.61 mU/mg in S1 and S2, respectively. The AChE activity did not vary with cancer, showing 2.46+/-0.45 mU/mg in S1 and 2.70+/-0.53 mU/mg in S2 from PC samples. Amphiphilic dimers and monomers and hydrophilic dimers of AChE were identified in BHP and PC tissues. Their contribution was affected by cancer with a great increase in hydrophilic dimers in the cancerous samples. Significant levels of both AChE and BChE activities were found in seminal fluid and homogenates from spermatozoids. Enzymatic activity dropped in samples with abnormal seminal parameters as sperm count and mobility.

Cholinergic system and cell proliferation.

The cholinergic system, comprising acetylcholine, the proteins responsible for acetylcholine synthesis and release, acetylcholine receptors and cholinesterases, is expressed by most human cell types. Acetylcholine is a neurotransmitter, but also a local signalling molecule which regulates basic cell functions, and cholinergic responses are involved in cell proliferation and apoptosis. So, activation of nicotinic and muscarinic receptors has a proliferative and anti-apoptotic effect in many cells. The content of choline acetyltransferase, acetylcholine receptors and cholinesterases is altered in many tumours, and cholinesterase content correlates with patient survival in some cancers. During apoptosis, acetylcholinesterase is induced and appears in the nuclei. Acetylcholinesterase participates in the regulation of cell proliferation and apoptosis through hydrolysis of acetylcholine and by other catalytic and non catalytic mechanisms, in a variant-specific manner. This review gathers information on the role of cholinergic system and specially acetylcholinesterase in cell proliferation and apoptosis.

Most Acetylcholinesterase Activity of Non-Nervous Tissues and Cells Arises from the AChE-H Transcript

While the functional implications of AChE-T, PRiMA and ColQ have been firmly established, those of glypiated AChE remain uncertain. Insights into the physiological meaning of glycosylphosphatidylinositol (GPI)-linked AChE-H were gained by comparing nervous and non-nervous tissues for the amount of AChE mRNA variants they contained. PCR showed that AChE-T mRNA prevailed in the mouse brain, spinal cord, sciatic nerve and muscle, and AChE-H mRNA in the bone marrow and thymus, as well as in the human gut. The similar levels of AChE-T and AChE-H mRNAs in mouse liver and human kidney contrasted with the almost exclusive presence of catalytically active AChE-H in both organs. The absence of PRiMA mRNA in liver suggested that the tetramers made of AChE-T fail to bind to the cell membrane and are secreted due to the lack of PRiMA in non-nervous organs. In contrast, glypiated AChE-H is largely and lastingly bound to the cell membrane. Thus, non-synaptic glypiated AChE-H seems to be the counterpart of synaptic PRiMA-linked AChE-T, the former designed for clearing ACh waves, the latter for confronting ACh bursts, and both for helping to protect cells against the harmful effects of durable nicotinic and muscarinic activation.

Butyrylcholinesterase activity and molecular components in thymus of healthy and merosin-deficient Lama2dy mice

The laminin-alpha2 chain, referred to as merosin, forms part of the laminin-2 heterotrimer (alpha2beta1gamma1), which is principally expressed in the basement membrane of muscle. Nearly half of patients suffering from congenital muscular dystrophy (CMD) have abnormalities in the laminin-alpha2 chain (LAMA2) gene, and the merosin-deficient Lama2dy mouse shows CMD. The expression of merosin in thymus, the abnormalities in the gland of Lama2dy mice, and the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in thymus prompted us to study the possible effects of the deficiency of merosin on thymus BuChE. We found that, while AChE activity decreased by approximately 50% in merosin-deficient thymus, the deficiency had little effect on BuChE activity. About 65% of thymus BuChE activity was extracted with a saline buffer and 30% with 1% Triton X-100. Sedimentation analyses and phenyl-agarose chromatography showed that thymus contained amphiphilic BuChE monomers (G(1)(A),44%) and dimers (G(2)(A),33%), and hydrophilic tetramers (G(4)(H),23%). Binding assays with various plant lectins revealed differences between the oligoglycans linked to BuChE tetramers and lighter components. The deficiency of merosin had no effect on the biosynthesis of thymus BuChE as judged by the lack of major changes between control and Lama2dy mice thymuses in the distribution of BuChE molecules and the level of lectin binding. The detoxifying action of BuChE, its role as a backup to AChE, and the relevance of the cholinergic dialogue between T cells and stromal cells for T lymphocyte proliferation, maturation and survival support a physiological function for BuChE in thymus.

Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma

Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p=0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p=0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients.