Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.     

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

The effects of calcium and strontium on the quantal content of nerve-evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2-0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca(2+)](o) was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca(2+)](o). Hence, extracellular calcium affected mainly the late, 'asynchronous', portion of phasic release. The average quantal content grew exponentially from 0.09 +/- 0.01 to 1.04 +/- 0.07 with the increase in [Ca(2+)](o) without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2-1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) (25 micromol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 micromol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms.     

Hair Mercury Association with Selenium,Serum Lipid Spectrum,and Gamma-Glutamyl Transferase Activity in Adults

The primary objective of the research is to estimate the dependence between hair mercury content, hair selenium, mercury-to-selenium ratio, serum lipid spectrum, and gamma-glutamyl transferase (GGT) activity in 63 adults (40 men and 23 women). Serum triglyceride (TG) concentration in the high-mercury group significantly exceeded the values obtained for low- and medium-mercury groups by 72 and 42 %, respectively. Serum GGT activity in the examinees from high-Hg group significantly exceeded the values of the first and the second groups by 75 and 28 %, respectively. Statistical analysis of the male sample revealed similar dependences. Surprisingly, no significant changes in the parameters analyzed were detected in the female sample. In all analyzed samples, hair mercury was not associated with hair selenium concentrations. Significant correlation between hair mercury content and serum TG concentration (r?=?0.531) and GGT activity (r?=?0.524) in the general sample of the examinees was detected. The respective correlations were observed in the male sample. Hair mercury-to-selenium ratios significantly correlated with body weight (r?=?0.310), body mass index (r?=?0.250), serum TG (r?=?0.389), atherogenic index (r?=?0.257), and GGT activity (r?=?0.393). The same correlations were observed in the male sample. Hg/Se ratio in women did not correlate with the analyzed parameters. Generally, the results of the current study show the following: (1) hair mercury is associated with serum TG concentration and GGT activity in men, (2) hair selenium content is not related to hair mercury concentration, and (3) mercury-to-selenium ratio correlates with lipid spectrum parameters and GGT activity.     

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.     

Differential regulation of sphingomyelin synthesis and catabolism in oligodendrocytes and neurons

Neurons (both primary cultures of 3-day rat hippocampal neurons and embryonic chick neurons) rapidly converted exogenous NBD-sphingomyelin (SM) to NBD-Cer but only slowly converted NBD-Cer to NBD-SM. This was confirmed by demonstrating low in vitro sphingomyelin synthase (SMS) and high sphingomyelinase (SMase) activity in neurons. Similar results were observed in a human neuroblastoma cell line (LA-N-5). In contrast, primary cultures of 3-day-old rat oligodendrocytes only slowly converted NBD-SM to NBD-Cer but rapidly converted NBD-Cer to NBD-SM. This difference was confirmed by high in vitro SMS and low SMase activity in neonatal rat oligodendrocytes. Similar results were observed in a human oligodendroglioma cell line. Mass-Spectrometric analyses confirmed that neurons had a low SM/Cer ratio of (1.5 : 1) whereas oligodendroglia had a high SM/Cer ratio (9 : 1). Differences were also confirmed by [³H]palmitate-labeling of ceramide, which was higher in neurons compared with oligodendrocytes. Stable transfection of human oligodendroglioma cells with neutral SMase, which enhanced the conversion of NBD-SM to NBD-Cer and increased cell death, whereas transfection with SMS1 or SMS2 enhanced conversion of NBD-Cer to NBD-SM and was somewhat protective against cell death. Thus, SMS rather than SMases may be more important for sphingolipid homeostasis in oligodendrocytes, whereas the reverse may be true for neurons.     

Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass

Attachment of oligodeoxynucleotides (ODNs) containing benzaldehyde (BAL) groups to semicarbazide-coated glass (SC-glass) slides is described. 5′-BAL-ODNs are prepared using automated DNA synthesis and an acetal-protected BAL phosphoramidite reagent. The hydrophobic protecting group simplifies purification of BAL-ODNs by reverse phase HPLC and is easily removed using standard acid treatment. The electrophilic BAL-ODNs are stable in solution, but react specifically with semicarbazide groups to give semicarbazone bonds. Glass slides were treated with a semicarbazide silane to give SC-glass. BAL-ODNs are coupled to the SC-glass surface by a simple one-step procedure that allows rapid, efficient and stable attachment. Hand-spotted arrays of BAL-ODNs were prepared to evaluate loading density and hybridization properties of immobilized probes. Hybridization to radiolabeled target strands shows that at least 30% of the coupled ODNs were available for hybridization at maximum immobilization density. The array was used to probe single nucleotide polymorphisms in synthetic DNA targets, and PCR products were correctly genotyped using the same macroarray. Application of this chemistry to manufacturing of DNA microarrays for sequence analysis is discussed.     

A comparison of sodium arsenite- and hyperthermia-induced stress responses and abnormal development in cultured postimplantation rat embryos.

Acute exposures to sodium arsenite (50 microM) were embryotoxic in day 10 rat embryos exposed in vitro. Sodium arsenite-induced embryotoxicity was characterized by decreased growth (crown-rump length, somite number, and embryo protein content) and abnormal development (hypoplastic prosencephalon, abnormal somites, and abnormal flexion of the tail). At embryotoxic exposures, sodium arsenite also induced the synthesis of three heat shock proteins (hsps), one of which is recognized by a monoclonal antibody specific for the heat-inducible hsp 72. In addition, sodium arsenite induced the accumulation of heat-inducible hsp 70 mRNA. Although the abnormal morphologies induced by sodium arsenite and hyperthermia appear to be different, the stress response as measured by the synthesis of hsps, the accumulation of hsp 72 protein, and the accumulation of hsp 70 mRNA is similar in embryos exposed to these two embryotoxic agents. Thus, sodium arsenite and hyperthermia both induce a stress response; however, the relationship between the induction of a stress response and the subsequent abnormal development that ensues is unclear.     

The effect of oxygen concentration on the teratogenicity of salicylate, niridazole, cyclophosphamide, and phosphoramide mustard in rat embryos in vitro

Comparisons were made of rat embryos cultured at 5% or 20% oxygen in the presence of salicylate (SAL), cyclophosphamide (CP), niridazole (NDZ), or phosphoramide mustard (PM). Multiple regression analyses were used to compare the effects of drug concentration, oxygen concentration, and the product of drug times oxygen concentration on malformation incidence, viability, and protein content of embryos cultured for 24 hours. Drug concentration significantly affected malformation incidence or severity and protein content (P less than 0.001) for the four drugs tested. Oxygen concentration significantly affected protein content for the four compounds (P less than 0.001) but affected malformation incidence only with NDZ. Furthermore, the interaction of oxygen concentration and drug concentration significantly affected the malformation incidence in the presence of NDZ (P less than 0.001), and protein content (P less than 0.001) and viability (P less than 0.001) in the presence of CP. The pattern of significant effects of the independent variables (drug concentration, oxygen concentration, and drug times oxygen concentration) is consistent with the hypotheses of oxygen-dependent metabolism (or lack of metabolism) of the drugs in question. NDZ, which is thought to be converted to reactive intermediates by an oxygen-inhibited nitroreductase, was more toxic at reduced oxygen tension. CP, which is activated by an oxygen-dependent P-450 system, was more toxic with increased oxygen tension. Significant effects of the independent variables on embryos exposed to SAL or PM were consistent with the effects on control embryos, notably, increased protein content with increased oxygen.