Characterization of Acetylcholinesterase Secretion in Neuronal Cultures and Regulation by High K+ and Soluble Factors from Target Cells

Abstract: We have observed that cultured neurons from chick spinal cord and the neuroblastoma hybrid line 108CC15 released lower amounts of acetylcholinesterase (AChE) when compared with the parental line, N18TG2. AChE activity extracted by hypotonic buffer, which can be regarded as the source of the released enzyme, was considerably higher in the parental than in the hybrid 108CC15 (respectively, ∼80% and ∼40% of cellular activity). On the other hand, evaluation of ectocellular, with respect to total, AChE activity showed that in N18TG2 cells only 7% of AChE was localized on the plasmalemma, whereas in the hybrid line the percentage of ectocellular activity was 3.7 times higher than in the parental line. We have also examined the effect of cytochalasin B and nocodazole. In the N18TG2 line, the former did not affect AChE release, which was significantly reduced by the latter. High K⁺ level in the culture medium, of both N18TG2 and hybrid 108CC15 cultures, induced an increase in AChE secretion; Ca²⁺ presence was required for high K⁺-induced release. Muscle extracts increased AChE secretion in both the hybrid 108CC15 and the spinal cord neurons. The present data suggest that AChE secretion during neuronal development is modulated by depolarizing stimuli and by soluble factors produced by target cells and may be involved in the control of neuronal differentiation.     

Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation

Background

Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.

Methods

Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca²⁺]_i) were conducted.

Results

Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca²⁺]_i revealed changes in membrane current in response to ACh and in [Ca²⁺]_i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca²⁺]_i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.

Conclusions

Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca²⁺-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.     

Studies of Sulfate Utilization by Algae. 6. Adenosine-3'-Phosphate-5'-Phosphosulfate (PAPS) as an Intermediate in Thiosulfate Formation From Sulfate by Cell-Free Extracts of Chlorella

When cell-free preparations of Chlorella pyrenoidosa Chick (Emerson strain 3) form thiosulfate from labeled sulfate, another radioactive compound also appears. This compound has been isolated in quantity and is shown to be identical with adenosine-3′-phosphate-5′-phosphosulfate (PAPS) on the basis of its chromatographic and electrophoretic behavior, chemical composition, sensitivity to selective degradative enzymes, and its ability to serve as a substrate for rat liver aryl sulphotransferase. In addition, as expected for PAPS, the compound on mild acid treatment yields all of its radioactive sulfur as sulfate, and is converted to a compound identical with adenosine-3′,5′-diphosphate (PAP). Replacement of sulfate and ATP by this PAP³⁵S in the usual incubation mixture yields the same product, thiosulfate, which can be isolated as such or detected as acid-volatile radioactivity. This conversion of PAP³⁵S to thiosulfate still requires the addition of Mg²⁺ and a reductant such as 2,3-dimercaptopropan-1-ol (BAL). The cause of our previous result that high concentrations of ATP inhibit thiosulfate formation from sulfate can be ascribed to a small amount of PAP contaminating the ATP preparations, since PAP proves to be an exceedingly effective inhibitor of the conversion of PAP³⁵S to thiosulfate. Sulfate reduction to thiosulfate by Chlorella extracts is discussed and compared with similar systems from other organisms.     

Origins of asexuality in <Emphasis Type="Italic">Bryobia</Emphasis>mites (Acari: Tetranychidae)

Background  

Obligate asexual reproduction is rare in the animal kingdom. Generally, asexuals are considered evolutionary dead ends that are unable to radiate. The phytophagous mite genus Bryobia contains a large number of asexual species. In this study, we investigate the origin and evolution of asexuality using samples from 111 populations in Europe, South Africa and the United States, belonging to eleven Bryobia species. We also examine intraspecific clonal diversity for one species, B. kissophila, by genotyping individuals from 61 different populations. Knowledge on the origin of asexuality and on clonal diversity can contribute to our understanding of the paradox of sex.     

CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation

Background  

Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.     

Local erythropoietin and endothelial progenitor cells improve regional cardiac function in acute myocardial infarction

Background

Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI.

Methods and Results

In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone.

Conclusion

Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.

     

Spatial and temporal expression of surfactant proteins in hyperoxia-induced neonatal rat lung injury

Background

Although personal cigarette smoking is the most important cause and modulator of chronic obstructive pulmonary disease (COPD), secondhand smoke (SHS) exposure could influence the course of the disease. Despite the importance of this question, the impact of SHS exposure on COPD health outcomes remains unknown.

Methods

We used data from two waves of a population-based multiwave U.S. cohort study of adults with COPD. 77 non-smoking respondents with a diagnosis of COPD completed direct SHS monitoring based on urine cotinine and a personal badge that measures nicotine. We evaluated the longitudinal impact of SHS exposure on validated measures of COPD severity, physical health status, quality of life (QOL), and dyspnea measured at one year follow-up.

Results

The highest level of SHS exposure, as measured by urine cotinine, was cross-sectionally associated with poorer COPD severity (mean score increment 4.7 pts; 95% CI 0.6 to 8.9) and dyspnea (1.0 pts; 95% CI 0.4 to 1.7) after controlling for covariates. In longitudinal analysis, the highest level of baseline cotinine was associated with worse COPD severity (4.7 points; 95% CI -0.1 to 9.4; p = 0.054), disease-specific QOL (2.9 pts; -0.16 to 5.9; p = 0.063), and dyspnea (0.9 pts; 95% CI 0.2 to 1.6 pts; p < 0.05), although the confidence intervals did not always exclude the no effect level.

Conclusion

Directly measured SHS exposure appears to adversely influence health outcomes in COPD, independent of personal smoking. Because SHS is a modifiable risk factor, clinicians should assess SHS exposure in their patients and counsel its avoidance. In public health terms, the effects of SHS exposure on this vulnerable subpopulation provide a further rationale for laws prohibiting public smoking.